Ann. Hum. Genet., Lmd. (1973), 37, 9 With 2 p l & ~ Printed in Great Britain

9

Banding patterns and autoradiographic studies of cells with an X-autosome translocation

BY MARY LUCAS Galton Laboratory, University College London

AND ALISON SMITHIES Central Middlesex Hospital

There are only a few reports of X-autosome translocations in man. In two cases (Mukherjee & Burdette, 1966; German, 1967) the translocated X was identified by autoradiography in cells from congenitally malformed children. In another abnormal child (Neuhauser & Back, 1967) the autoradiographic findings suggested that an X-autosome translocation was present. In two other cases (Mann et al. 1965; Thorburn, Martin & Pathak, 1970) possible X-autosome translocations were found in phenotypically normal females presenting with primary amenor- rhoea.

Recent techniques using special staining to produce banding patterns allow precise identi- fication of chromosomes. Two X-autosome translocations (Buckton et al. 1971; Ruddle et al. 1971) have been identified with these methods in phenotypically normal patients, some of whom were fertile. This paper reports the banding patterns and autoradiographic findings in cells with an X-autosome translocation from a patient who presented with ovarian failure shown by primary amenorrhoea and lack of secondary sexual development. No other phenotypic abnormalities were detected in this patient.

FAMILY STUDIES

The proposita is one of a family of nine. Her father is dead. Specimens were obtained from her mother and seven of her siblings, all of whom showed a normal karyotype. Dr Sanger of the Lister Institute found that all the available specimens were Xg(a+). The results of blood grouping and enzyme studies are available on request (number MRC 1798).

MATERIALS AND METHODS

Buccal smears were stained with lacto-aceto-orcein and permanent preparations were stained with cresyl violet.

Chromosome preparations were made from peripheral blood cultures. Some slides were stained with aceto-orcein and some by an adaptation of the trypsinization and Giemsa method of Seabright (1971) as previously described (Lucas et al. 1972).

A second blood culture was used for autoradiography . Tritiated thymidine (specific activity 21.9 c./mmol.) was added a t a concentration of 0.2 pc./ml. 6 hr. before harvesting and 4 hr. before the addition of colcemid. The well-spread mitoses were photographed before stripping film (Kodak AR 10) was applied to the slides and exposed for 1 week. After development of the film in D 19 for 6 min. the cells were relocated and re-photographed if some, but not all, of the chromosomes were labelled.

1’0 MARY LUCAS AND ALISON SMITHIES

19 20 21 22

Fig. 1. Banding patterns. 46,X, t ( X ; 15)(g13;~13) .

RESULTS

Barr bodies 6f normal size were present in the nuclei of buccal mucosa cells. Nuclei containing small Barr bodies or two Barr bodies were not found.

Chromosome preparations stained with aceto-orcein showed that the chromosome number was 46, but the karyotype was not normal. There were five instead of six D chromosomes and apparently an extra E chromosome. There were 15 normal C chromosomes and an abnormal chromosome of the same size as the larger C chromosomes. This chromosome was easily identified in every cell by the marked secondary constriction close to the centromere. In many cells there was association between the satellite region of an acrocentric and this constriction.

After special staining (Fig. I} one of the ‘E’ chromosomes showed the banding pattern characteristic of the short arms of the X . The abnormal chromosome showed the pattern of a no. 15, with the long arms of the X distal to the secondary constriction. This karyotype therefore has a translocation between a 15 and an X chromosome; 46,X, t (X;15) (q13;p13) .

The cells labelled with tritiated thymidine could be assigned to one of three groups: (A) Eighteen cells with few grains showed only one labelled chromosome and in every cell

this was a normal member of group C. This pattern is seen in cells from a person with a normal

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X-autosorne translocation 11

female karyotype in which one X is late replicating. The finding that the normal X was late replicating was confirmed by Dr Eva Sujansky, Dr Lillian Y. Hsu and Dr K. Hirschhorn of the Mount Sinai School of Medicine.

(B) In 24 cells showing more than one labelled chromosome, one normal C was heavily labelled and the abnormal chromosome showed grains over the long arms distal to the secondary constriction. In five cells these were the only labelled parts. In the other 19 cells in this group other chromosomes were labelled so that it was possible to distinguish between nos. 4 and 5 and to identify the D chromosomes, of which four were ‘hot’ (pairs nos. 13 and 14) and one was ‘cold’ (no. 15). In some of these cells two E chromosomes were labelled (pair no. 18) (Plate I, Fig. 2).

(C) The remaining 28 cells also showed labelling of some autosomes but differed from the preceding category in that three ‘E’ chromosomes were ‘hot’. In cells from a person with a normal karyotype, one pair (no. 17) is early-replicating and one pair (no. 18) is late-replicating, so it is assumed that in these cells the third ‘hot E ’ is the deleted X ( X p ) . These cells also showed only one unlabelled D chromosome (Fig. 3).

DISCUSSION

The autoradiography showed that only one D was early-replicating, thus confirming the findings from the Giemsa staining that a no. 15 had taken part in the translocation. This translocated no. 15 retained its property of early replication although attached to part of an X which replicated much later in the cycle.

The autoradiography indicated that the replication of the X chromosomal material was in the order: X p probably before nos. 4, 13, 14 and 18, X q probably later than 4, 13, 14 and 18 and just before the normal X , which is late-replicating.

Two workers, Schmid (1963) and Lubs quoted by Miller (1970), have reported that the ‘cold’ (isocyclic) X of a normal karyotype can sometimes be identified by comparison with the other C chromosomes. They identified it as the one chromosome of the C group having heavy diffuse labelling over the entire long arms and labelled over the short arms, except in cells labelled during the very late stages of replication. The labelling of the abnormal X in this patient appears to agree with these findings, although it is not possible to compare findings from cells with two normal X chromosomes with the results obtained here where one of the X’s is in two parts.

Previous studies of patients with an abnormal amount of X chromosomal material have shown that there is a correlation between the size of the Barr bodies and the size of the abnormal X , which is the late-replicating X . In this case, the finding of normal-sized Barr bodies correlates with the observation that the latest-replicating chromosome is an apparently normal group C chromosome. In the family with an X/14 translocation reported by Buckton et al. (1971), all the members who carried the balanced translocation and therefore had a normal amount of X chromosomal material showed Barr bodies of the normal size and the normal X to be late-replicating.

In the families reported by Buckton et al. (1971) and Ruddle et al. (1971) the balanced translocation carriers were fertile. It is therefore not possible to say whether the finding of a translocation involving the X in this patient is related to the ovarian function.

12 MARY LUCAS AND ALISON SMITHIES As no other members of the family show a translocation, it is very likely that the rearrange-

ment in this patient occurred during gametogenesis in one parent or during a very early division in the zygote.

SUMMARY

A patien6 presenting with ovarian failure was found to have a translocation between a group D and a group C chromosome, shown by special staining to be a balanced XI15 trans- location.

This rearrangement was not present in any other members of the family studied. Autoradiographic studies showed that the normal X was the late-replicating X and suggested

that the short arms of the abnormal X replicated before nos. 4, 13, 14 and 18 and that the long arms of the abnormal X replicated after nos. 4, 13, 14 and 18 and just before the normal late-replicating X.

We are grateful to Mr H. H. Thomson of the Central Middlesex Hospital for permission to describe the patient under his care and to Dr de la Chapelle of the Folkhalsan Institute of Genetics, Helsinki, and to Dr Lindsten of the Karolinska Sjukhuset, Stockholm, for collecting the specimens for the family studies.

REFERENCES

BUCKTON, K. E., JACOBS, P. A., RAE, L. A., NEWTON, M. S. & SANGER, R. (1971). An inherited X-autosome translocation in man. Ann. Hum. Genet. 35, 171.

GERMAN, J. (1967). Autoradiographic studies of human chromosomes. I. A review. Proc. 3rd Int. Gong. Hum. Genet. (ed. J. F. Crow and J. V. Neel), p. 123.

LTJCAS, M., WALLACE, I. & HIRSCHHORN, K. (1972). Recurrent abortions and chromosome abnormalities. J . Obstet. Gynaec. Br. Commonw. 79, 1119.

MA", J. D., VALDMANIS, A., CAPPS, S. C. & PUITE, R. H. (1965). A case of primary amenorrhea with a translocation involving chromosomes of Groups B and C. A m . J . Hum. Genet. 17, 377.

MILLER, 0. J. (1970). Autoradiography in human cytogenetics. In Advances in Human Genetics, vol. I (ed. H. Harris and K. Hirschhorn). Plenum Press.

MUKHERJEE, D. & BURDETTE, W. J. (1966). Multiple congenital abnormalities associated with a ring 3 chromosome and translocated 3/X chromosome. Nature, Lond. 212, 153.

NEUHAUSER, G. & BACK, F. (1967). X-Autosom-Translokation bei einem Kind mit multiplen Mizbildungeil. Humangenetik 3, 300.

RUDDLE, F. H., CHAPMAN, V. M., RICCIUTI, F., MURNANE, M., KLEBE, R. & MEERA KHAN, P. (1971). Linkage relationships of seventeen human gene loci as determined by man-mouse somatic cell hybrids. Nature New Biol. 232, 69.

SCHMID, W. (1963). DNA replication patterns of human chromosomes. Cytogenetics 2, 175. SEABRIGHT, M. (1971). A rapid banding technique for human chromosomes. Lancet ii, 971. THORBURN, M. J., MARTIN, P. A. & PATHAK, U. N. (1970). Possible X/autosomal translocation in a girl

with gonadal dysgenesis. J . Med. Genet. 7 , 402.