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
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.
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
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.
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
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
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.
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
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
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).