Chromosomal Autoradiography in the Cri du Chat Syndrome

J. GERMAN2, J. LEJEUNE3, M. N. MACINTYRE4 and J. DE GROUCHY5.

Cytogenetics 3: 347-352 (1964).


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Introduction

The four long, sub-acrocentric chromosomes constituting Group 4-5 of the human complement cannot by morphological features alone be distinguished with certainty from one another (GERMAN, 1964a). Nevertheless, the group is particularly suitable for autoradiographic analysis after the DNA has been labeled with H3-thymidine, because two of its members show extensive replication throughout their length late in the S-period (GERMAN, 1964a, b). The other two members are easily distinguished from the two late-replicators because characteristically DNA synthesis is completed relatively early throughout much of the length of their long arms while it continues in their short arms.

The aberrant member of Group 4-5 in the cri du chat syndrome has been called a No. 5 by the discoverers of this disorder, LEJEUNE et al (1963). The appearance of this aberrant chromosome in cells from the six patients reported (BÖÖK et al., 1963; DE GROUCHY et al., 1964; LEJEUNE et al., 1963 ; LEJEUNE et al., 1964a, b) has been similar and the similar clinical abnormalities in each instance of the syndrome suggest the absence of approximately the same genetic material in each case. The magnitude of the deletion may vary somewhat from case to case. This variation, however, is beyond the limits of accurate measurement at the present tune. Six further cases are known by way of personal communications (C. E. BLANK; R. CEPPELLINI; P. E. CONEN; J. C. DE ALMEIDA; W. J. MELLMANN; M. MIKKELSEN; H. H. PUNNETT, 1963 and 1964), and the available data are in good agreement with the foregoing statement. Consequently autoradiographie study of the replicational sequence in the chromosomes of Group 4-5 in this syndrome was undertaken to determine whether the No. 5 is the earlier or the later replicating member of the group and whether the same autoradiographie pattern in the abnormal unit exists in cases of the syndrome from two different families.

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Methods and Clinical Data

Cells from venous blond were culturel in the presence of phytohemagglutinin for three days. Six hours before their fixation H3-thymidine was added to the culture medium, and three hours before fixation colcemid was added; this schedule has been found suitable for labeling the chromosomal regions which replicate during the final intervals of the S-period. The concentration of isotope, method of handling the chromosomes, autoradiographic analyses, and duplicate photography technique were those described by GERMAN (1964b). The blood cells from Patient No. 1 were cultured and labeled in Paris, and slides were mailed to New York City; the blood from Patient No. 2 was mailed by air from Cleveland to New York City where nucleated cells were cultured and labeled. Autoradiography was performed simultancously on the slide preparations from both.

Patient No. 1 is an individual described in detail elsewhere (case No. 1 of LEJEUNE et al., 1963, 1964b). She is eighteen months old and exhibits the typical features of the syndrome: mental retardation, hypertelorism and epicanthal folds, dermatoglyphic anomalies, and a cry similar to that of a cat. Patient No. 2 is seven months old and is being described in an extensive repart by MACINTYRE et al. (1964). Her major abnormalities are severe mental and growth retardation, microcephaly, a rounded face, hypertelorism, oblique palpebral fissures, epicanthal folds, low set ears, micrognathia, and the characteristic cry.

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Results

The chromosomal complements of the two patients were similar and included one abnormal member of Croup 4-5 and 45 other chromosomes of normal appearance. In Group 4-5 the abnormal chromosome had proportions comparable to that reported by LEJEUNE et al. (1963, 1964a, b). Its long arm resembled that of the other normal members of the group, but the short arm was greatly decreased in length, indicating a major deletion.

The study is comprised of a total of 42 radioactive tells in metaphase. The distribution of grains over the chromosomes were examined microscopically, and the chromosomes were identified by comparison with the preliminary photographs of the cells. Photographs of seven cells derived from Patient No. 2 and six from Patient No. 2 were cut and analysed by the duplicate photography technique, as shown in the figure. The cells derived from each patient showed the same patterns. In every informative cell, the chromosome with the deletion was one of the earlier replicators of Group 4-5, and had a degree of radioactivity in its long arm comparable to another relatively lightly labeled member of the group. Must of the segment of the short arm which replicates later than the long arm was absent. The remaining two non-deleted members, the Nos. 4, had relatively heavy and extensive radioactivity throughout their lengths. Thus, except for the absence of grains due to the missing portion of a No. 5 chromosome, no alteration in the expected autoradiographic pattern in the chromosomes of Group 4-5 was detected. In the Figure the pattern of radioactivity in normal cells labeled late in the S-period is demonstrated, for comparison with Chat in cells from the patients with the cri du chat syndrome.

In the autoradiographs of the remaining chromosomes of the cells from the two patients the characteristic late synthesis in an X-chromosome, in two of Group 13-15, and in the Nos. 16 and 18 was observed. The usual occasional examples of autosomal homologue asynchrony of synthesis were present.


Fig. The chromosomes of Group 4-5 and their autoradiographs in cells from normal individuals and from the cri du chat syndrome. Cells I-IV: blood cells from three different normal individuals. Cell V : cultured fibroblast from skin of a normal individual. (In this tell the similarity of the autoradiographic patterns in fibroblasts and blood cells is demonstrated indicating that cells derived from various tissues of patients with chromosomal abnormalities in Group 4-5 can be informative.) Cells VI-IX: blood cells from Patient No. 1. Cells X-XII: blood cells from Patient No. 2. Two chromosomes, the Nos. 4., normally continue extensive DNA synthesis during the terminal intervals of the S-period. The abnormal chromosome in the cri du chat syndrome, a No. 5, identifies itself in these autoradiographs not as a late replicator but as one of the pair which normally completes synthesis early in the long arms, relatively late in the short arms. Most of the late-replicating region of its short arm has been deleted. Reproduced at 1,730 x except Cell IX which is at 1,870 x.

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Discussion

The similarity of the clinical features in various individuals with the cri du chat syndrome suggests that a deletion in the same chromosome has occurred in each instance. Furthermore, the abnormal chromosome in each of the two patients in the present study exhibited the same autoradiographie pattern. This chromosome is one of the pair of Group 4-5 in which replication in the long arms is completed relatively early and which has already tentatively heen referred to as the No. 5 pair (GERMAN, 1964a). In each cell studied by autoradiography, the long arm of one other non-deleted unit of the group displayed a similar pattern of radioactivity. In normal cells, there is usually a moderately good synchronization of replication in the two members of a recognizable autosomal pair. Since the abnormal chromosome of the cri du chat syndrome has been referred to as a No. 5 (LEJEUNE et al., 1963), it may be concluded that the No. 5 chromosomal pair is the pair characterized by relatively early completion of DNA synthesis in the long arms and by later synthesis in the short arms. The remaining two chromosomes of the group, the Nos. 4, then may be defined as Chose which replicate late throughout their lengths.

From an earlier study (GERMAN, 1964a) it became apparent that from structural features atone many chromosomes of the human complement would not, by cytological techniques now commonly employed, be distinguishable. For example, in Group 4-5 the Nos. 4. (i.e., the lace replicating pair) have a mean length almost identical to that of the Nos. 5. Nor is the arm ratio more useful, since the mean of the Nos. 4. is 2.57 while that of the Nos. 5 is 2.64, and in only 29 % of cells is the arm ratio of both Nos. 4 less than that of both Nos. 5. However, many if not all chromosomes of the complement of the human lymphocyte display a characteristic and distinguishing DNA replicational pattern. Fortuitous circumstances such as those in the present study of the cri du chat syndrome may eventually lead to definition of many or all of the chromosomes with structurally non-distinctive characteristics. In turn, the autoradiographic pattern will be useful in determining which units in various newly discovered chromosomal abnormalities have participated in a rearrangement or a numerical deviation. Furthermore, it may be possible to determine whether the same member of the complement is affected in different patients, the chromosomes of which show similar abnormalities when assayed by morphology alone.

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Conclusions

In the cri du chat syndrome the abnormal chromosome, a No. 5, is one of the pair in Group 4-5 characterized by relatively early completion of DNA synthesis in the long arms. The two remaining members of this group, characterized by extensive late replication throughout their lengths, may now therefore be recognized as the Nos. 4.


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References

BÖÖK, J. A.; ATKINS, L. and SANTESSON, B.: Some new data on autosomal aberrations in man. Path. Biol. II: 1159-1162 (1963).

DE GROUCHY, J.; ARTHUIS, M.; SALMON, C.; LAMY, M. and THIEFFRY, S: Le syndrome du cri du chat, une nouvelle observation. Ann. Génét. 7: 13-16 (1964).

GERMAN, J.: Identification and characterization of human chromosomes by DNA replication sequence. In: Symposia of the International Society for Cell Biology, vol. 3 (ed. HARRIS, R. J. C.), pp. 191-2O7 (Academic Press, New York 1964a).

GERMAN, J.: The pattern of DNA synthesis in the chromosomes of human blood cells. J. cell Biol. 20: 37-55 (1964b).

LEJEUNE, J.; LAFOURCADE, J.; BERGER, R.; VIALETTE, J.; BOESWILLWALD, M.; SERINGE, P. and TURPIN, R.: Trois cas de délétion partielle des bras courts d'un chromosome 5. C. R. Acad. Sci. 257: 3098-31O2 (1963).

LEJEUNE, J.; GAUTIER, M.; LAFOURCADE, J.; BERGER, R. and TURPIN, R.: Délétion partielle du bras court du chromosome 5. Cinquième cas de syndrome du cri du chat: Ann. Génét. 7: 7-1O (1964a).

LEJEUNE, J.; LAFOURCADE, J.; DE GROUCHY, J.; BERGER, R.; GAUTIER, MARTHE; SALMON, C. and TURPIN, R.: Délétion partielle du bras court du chromosome 5. Individualisation d'un nouvel état morbide. Semaine Hôpitaux Paris 40: 1169-1179 (1964b).

MACINTYRE, M. N.; STAPLES, W. .I.; LAPOLLA, J. J. and HEMPEL, J. M.: The "cat cry" syndrome. Amer. J. Dis. Child. 108: 538-542 (1964).