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Introduction
Since the original description in 1963 of the chromosomal anomaly 5p-
(cri du chat syndrome) by Lejeune et al., over 300 cases have been reported
(Niebuhr, 1978 ; Rethoré, 1977). This condition results from deletion of 15-80
% of the short arm of chromosome 5 ; bands 5p14 and 5p15 are missing in all
cases. The dysmorphic syndrome is characterized by severe mental retardation,
failure to thrive and hypotonia at birth, as well as by the catlike cry which
gave the syndrome its name. Little is known about the metabolic consequences of
deletion of this part of the short arm of chromosome 5 and, to date, no genes
have been mapped to this region.
A general hypothesis has been proposed (Lejeune, 1983), suggesting
that a defect in monocarbon metabolism and in related metabolic pathways
(purine and pyrimidine synthesis and methylation reactions) could be of great
importance in mental retardation with or without neurological deterioration
and/or psychiatric complications. In the present work, we investigated
particularly the purine pathways, searching for defects, possible causes and
consequences thereof which could constitute a basis for reflection concerning a
general mechanism of mental impairment.
Within the context of metabolic changes secondary to a primary lesion,
it should be possible to unveil the pathophysiological consequences of a
chromosomal imbalance, as Garrod had already suggested in 1908 for inborn
errors of metabolism.
To test this hypothesis, we designed a simple research protocol
whereby lymphocytes from patients with different chromosomal anomalies or
suffering from mental retardation syndromes of unknown etiology, are cultured
in the presence of different inhibitors of purine synthesis, as well as in the
presence of exogenous nucleotides (fig. 1).
Changes in the in vitro sensitivity to these additives are examined by
calculating changes in the mitotic index and by comparing these fluctuations to
those observed in the patient's own control culture, as well as to those
observed in normal controls or in patients suffering from other pathologies
than the one being currently studied.
 Fig. 1 - Purine
synthesis. Site of action of antimetabilites is marked by * (alanosine,
aminopterin, azaserine, mycophenolic acid)
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Material and methods
Sixteen peripheral blood samples from a total of twelve patients with
cri du chat, 5p- syndrome, were cultured for 72 hours in TC199 medium (Seromed)
supplemented with 25 % human AB serum, phyto-hemagglutinin -C (IBF France),
penicillin and streptomycin. Culture technique, harvesting and microscopic
examination were carried out according to the previously described method
(Lejeune et al., 1986 ; Peeters et al., 1989). Patient characteristics are
shown in table I.
The following products were used on initiation of the culture (all
products were Sigma products unless otherwise specified) :
1) inosine: 125 mg/l;
2) L-alanosine : 0.0625 mg/1 (kindly provided for by the Laboratory of
Biochemical Pharmacology, National Institute of Health, Bethesda, Maryland) ;
3) azaserine : 0.0156 mg/l;
4) adenosine : 16 mg/l ;
5) HAT medium: 62.5 µM hypoxanthine, 0.25 µM aminopterin, 10 µM
thymidine ;
6) cytidine: 6.25 mg/l ;
7) guanosine : 3.1 mg/l;
8) putrescine : 6.25 mg/l.
The following products were added 12-16 hours prior to termination of
the culture
9) mycophenolic acid: 0.6 x 10-6M ; 10) theophylline : 750 mg/l.
The entire panel could often not be performed for each patient due to
inadequate blood samples.
Ten patients were on no medication (except folic acid in three
patients and anti-convulsive therapy in one patient) at the time of the
lymphocyte culture ; six patients were on folinic acid, four of whom also
received inosine 50 mg/kg.
Controls used for the analysis were as follows: 136 normal adults, 91
patients with documented trisomy 21, 30 patients with known Xqfra syndrome and
83 patients with mental retardation (with or without a chromosomal anomaly
other than the ones mentionned above).
TABLE I. - Patient characteristics and variation of the
mitotic index in the presence of the two additives which were found to
significantly alter the mitotic index of the cri du chat patients studied.
| Name | Sex | Age | Karyotype | IQ | Treatment | ALAN | HAT |
1 | HD | F | 5 | 46,XX,del(5)(qter
p14.2:) | 38 | none | 83 % | - 97 % |
2 | BA | M | 23 | 43,XY,del(5)(qter
p14:) | | folinate | 16 % | - 69 % |
3 | HH | F | 30
| 46,XX,del(5)(qter p14:) | 60 | none | 32
% | |
4 | DP | M | 22
| 46,XY,del(5)(qter p14:) | 23 | folate | 19
% | |
5 | BI | F | 9
| 46,XX,del(5)(qter p14:) | 43 | folate | 40 %
| - 98 % |
6 | DJL | M | 24
| 46,XY,del(5)(qter p14:) | | none | 24 %
| |
7 | CC | F | 3 | 46,XX,del(5)(qter
p?:) | 44 | none | 8 % | - 68 %* |
8 | CC | F | 9
| 46,XX,del(5)(qter p13.3) | 33 | none | 16
% | |
9 | DE | F | 2
| 46,XX,del(5)(qter p14:) | 63 | folinate | - 7 %
| - 98 % |
10 | BN | M | 16 | 46,XY,r(5)
| | anti-conv thrapy | 4 % | - 98 % |
11 | SCH | F | 30
| 46,XX,del(5)(qter p33.3:) | 30 | none | 6
% | |
12 | CM | F | 6
| 46,XX,del(5)(qter p?:) | 45 | none | 6%
| - 85 % |
13 | | | | Same as 5
| | folinate + inosine | - 36 % | - 74 |
14 | | | | Same as 10
| | folinate + inosine | - 17 % | - 52 % |
15 | | | | Same 11
| | folinate + inosine | - 13 % | - 72 % |
16 | | | | Same as 12
| | folinate + inosine | - 29 % | - 73 % |
* Patient had stopped receiving folinic acid only
10 days prior to the blood lymphocyte culture. |
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Statistical analysis
Results were analyzed by comparing the mitotic index of each
experiment to the patient's own control culture and results were expressed as
the percentage increment or decrease in mitotic index. Statistical comparisons
between groups were based on Student's t-test.
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Results
As is shown in table II (only data relevant to the present study are
shown) 4/6 (67 %) patients with cri du chat syndrome, who were not receiving
inosine at the time of the blood lymphocyte culture, showed two significant
differences in their responses to the different products added on initiation of
the culture :
- a highly significant increase in mitotic index was observed in 5p-
lymphocyte cultures to which a low dose of alanosine had been added ;
- a significantly greater sensitivity to HAT medium was observed in
lymphocyte cultures of patents with cri du chat syndrome. There was a slightly
greater increment of the mitotic index in cultures to which either inosine or
putrescin had been added but this did not reach a 5 % significance level.
There was no significant difference additives.
The association : increase in mitotic index in the presence of low
dose L-alanosine and almost complete inhibition of the mitotic index in the
presence of HAT medium seems an almost specific association for patients with
cri du chat syndrome. To examine the specificity of this association we
effected a computer search looking for patients with an increase in mitotic
index of over 4 % in the presence of alanosine and a greater than 85 % decrease
in the presence of HAT medium. This association was found in: 16/362 (4.4 %)
patients from our total sample (patients with 5p- syndrome were excluded), in
4/136 (2.9 %) normal controls, in 1/91 (1 %) patients with trisomy 21, in 2/30
(6.6 %) patients with Xqfra syndrome and in 7/83 (8.4 %) patients with mental
retardation (4 of whom had a chromosomal anomaly: 4p-, 18q-, tri11qter,
tetrasomy 15qprox). It is noteworthy that all these patients had other
distinctive, discriminatory identifiable metabolic features. Because sample
sizes were too small to allow valid analysis work is currently in progress to
further characterize these patients.
As is shown in table III, (only data pertinent to this study are
shown), patients with cri du chat syndrome who were treated with inosine showed
a significant decrease in mitotic index in the presence of low dose alanosine,
decrease that was significantly greater than the one observed in normal
controls (0.05 > p > 0.025). In 5p- patients receiving treatment (either
folinic acid or folinic acid with inosine) the decrease in the mitotic index in
the presence of HAT medium was identical to the one observed in all the other
categories of patients studied.
Thus patients with cri du chat syndrome demonstrate, as shown in table
IV, complete reversal of their reaction to alanosine when given oral inosine.
Treatment with inosine and folinic acid cause " normalization " of their in
vitro sensitivity to HAT medium. These changes observe in vitro were highly
significant.
TABLE II. - Variations in the mitotic index observed in the
presence of exogenous inosine, L--alanosine, HAT medium and putrescine.
Comparisons and Student's t-test are made between cri du chat syndrome patients
not receiving inosine and either normal controls or patients presenting mental
retardation of another etiology.
| Additives |
Inosine | Alanos | HAT | Putrecine |
5p- not receiving inosine |
n | 12 | 12 | 7 | 6 |
m | 10,9 | 20,6 | - 88 | 20,8 |
s | 21,7 | 22,5 | 12,8 | 13,7 |
Total
sample | n | 295 | 246 | 166 | 135 |
m | 2,3 | 0,9 | - 66 | 3,4 |
s | 23,4 | 24,2 | 25,7 | 21,9 |
t | 1,2 | 2,8** | 2,2* | 1,9 |
Normal
controls | n | 100 | 76 | 40 | 19 |
m | 2,5 | - 0,8 | - 63 | 1,3 |
s | 22,5 | 21,5 | 26,9 | 16,9 |
t | 1,2 | 3,2*** | 2,4* | 2,5* |
Mental
retardation | n | 76 | 62 | 64 | 58 |
m | 2.8 | 1.1 | - 69 | 6.8 |
s | 24.1 | 24.6 | 24.6 | 24.1 |
t | 1.1 | 2.5** | 2* | 1.4 |
Trisomy
21 | n | 59 | 50 | 29 | 27 |
m | 0,5 | - 1,4 | - 59 | -3,3 |
s | 27 | 25.4 | 22.7 | 20.4 |
t | 1.2 | 2.7** | 3.2*** | 2.7** |
Xqfra
syndrome | n | 24 | 26 | 14 | 16 |
m | 1.2 | 3.4 | - 63 | - 2.4 |
s | 18.3 | 26.5 | 24 | 19.5 |
* p = 0.05, ** p
= 0.01, *** p =
0.005 |
TABLE III. - Variations in the mitotic index observed in the
presence of exogenous inosine, L--alanosine, HAT medium and putrescine.
Comparisons and Student's t-test are made between cri du chat syndrome patients
receiving inosine and either normal controls or patients presenting mental
retardation of another etiology.
| Additives |
Inosine | Alanos | HAT | Putrecine |
5p- not receiving inosine |
n | 4 | 4 | 4 | 3 |
m | -6 | -24 | -68 | 6 |
s | 7.4 | 9.2 | 9.1 | 16.3 |
Total
sample | n | 295 | 246 | 166 | 135 |
m | 2,3 | 0,9 | - 66 | 3,4 |
s | 23,4 | 24,2 | 25,7 | 21,9 |
t | 0.7 | 2* | 0.2 | 0.2 |
Normal
controls | n | 100 | 76 | 40 | 19 |
m | 2,5 | - 0,8 | - 63 | 1,3 |
s | 22,5 | 21,5 | 26,9 | 16,9 |
t | 0.8 | 2.1* | 0.4 | 0.4 |
Mental
retardation | n | 76 | 62 | 64 | 58 |
m | 2.8 | 1.1 | - 69 | 6.8 |
s | 24.1 | 24.6 | 24.6 | 24.1 |
t | 0.7 | 2* | 0.1 | 0.1 |
Trisomy
21 | n | 59 | 50 | 29 | 27 |
m | 0,5 | - 1,4 | - 59 | -3,3 |
s | 27 | 25.4 | 22.7 | 20.4 |
t | 0.5 | 1.7 | 0.8 | 0.7 |
Xqfra
syndrome | n | 24 | 26 | 14 | 16 |
m | 1.2 | 3.4 | - 63 | - 2.4 |
s | 0.8 | 2* | 0.4 | 0.7 |
* p = 0.05 |
TABLE IV. - Variation of the mitotic index in the presence of
different additives between cri du chat patients receiving: inosine plus
folinate or folinate only compared to patients not treated or receiving only
folic acid.
| Additives |
Inosine | Alanos | Azas | Adénosine | HAT | Putrecine |
5p- receiving inosine + folinate |
n | 4 | 4 | 4 | 4 | 4 | 2 |
m | -6 | -24 | 2 | -3 | -68 | 10 |
s | 7.4 | 9.2 | 12.5 | 7.6 | 9.1 | 5.6 |
5p- receiving no
inosine | n | 12 | 12 | 10 | 11 | 7 | 6 |
m | 10.9 | 20.6 | 1 | 10.9 | -88 | 20.9 |
s | 21.7 | 22.5 | 18 | 26.2 | 12.8 | 13.7 |
t | 1.5 | 3.6*** | 0.1 | 0.9 | 2.5* | 1 |
5p- receiving inosine + folinate or folinate
only | n | 6 | 6 | 6 | 6 | 6 | 2 |
m | -1.2 | -14.3 | -0.2 | 13.3 | -73 | 1.7 |
s | 10.4 | 18.3 | 12.7 | 18.8 | 14.7 | 14 |
5p- receiving no
treatment | n | 10 | 10 | 8 | 9 | 5 | 6 |
m | 11.3 | 23.8 | 1.8 | 13.5 | -89.2 | 20.8 |
s | 25 | 24 | 20.4 | 29.6 | 13 | 14.9 |
t | 1.2 | 3.3*** | 0.2 | 0 | 1.9 | 1.6 |
* p = 0.05, ** p
= 0.01, *** p =
0.005 |
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Discussion
In discussing these findings, a brief reminder of the metabolic sites
of action of the two products found to significantly alter the mitotic index of
5p- lymphocytes need be reviewed. In HAT medium (hypoxanthine, aminopterin,
thymidine), aminopterin is an inhibitor of dihydrofolate reductase, by virtue
of which, de novo purine and thymidine monophosphate synthesis is rendered
impossible. Hypoxanthine and thymidine are added to the medium to allow for
necessary cell rescue. A highly significant decrease in mitotic index of cri du
chat lymphocytes in the presence of HAT medium could therefore imply that 5p-
cells have some metabolic impediment in de novo nucleotide synthetis. This
defect could be :
- primary, i.e. an enzymatic defect due to the loss of genes coding
for de novo nucleotide synthesis pathway, enzymes which would be found in the
zone 5p14-5p15 ;
- or secondary, i.e. an enzymatic defect of necessary cofactors, for
example, folinic acid, needed for de novo nucleotide synthesis. The
normalization of the reaction to the toxic effect of HAT medium in 5p- patients
receiving folinic acid (with or without inosine) confirms this sensitivity but
does not allow us to answer this question satisfactorily and draw a defnite
conclusion.
L-alanosine interacts with the enzymes responsible for the metabolism
of the dicarboxylic aminoacids (L- acid and L-glutamic acid) and their amines
(Anandaraj et al., 1980). It is relevant that L-alanosine even at very low
concentrations, rapidly inhibits the conversion of IMP to AMP without affecting
pyrimidine nucleotide synthesis (Graff et al., 1976). L-ala- appears to be
converted to an inhibitory metabolite that reversibly inhibits adenylosuccinate
synthetase with consequent IMP accumulation in treated cells (Tyagi et al.,
1984).
In 5p- lymphocytes stimulated to divide actively, low dose L-alanosine
increased the mitotic index. This unexpected finding (antimetabolites generally
decrease the mitotic index) remains to be explained.
We hypothesize that lymphocytes from cri du chat patients benefit from
the IMP accumulation observed in the presence of L-alanosine. The slight
increase in mitotic index in the presence of exogenous inosine added to the
lymphocyte culture would be in keeping with this hypothesis. The
adenylosuccinate synthethase inhibition known to occur with alanosine could, in
our culture system, be by-passed by the large amounts of adenosine found in
TC199 medium. We therefore suggest that deletion of the terminal portion of the
short arm of chromosome 5 could be responsible for a defect in de novo purine
synthesis.
This hypothesis would be in keeping with the above mentioned severe
HAT toxicity observed in these patients. Indeed partial inhibition of de nova
purine synthesis would be particularly toxic in lymphocytes already partially
hampered along this metabolic pathway.
Furthermore, we have recently shown that in patients with cri du chat
syndrome there is a highly significant excess amount of asparagine and
aspartate in both serum and urine (Lejeune et al., 1990). Such an excess could
occur if one of the metabolic pathways of aspartate was impaired, especially if
its incorporation into purine de novo synthesis was hampered.
Taking into consideration these three findings it seems legitimate to
suggest that either phosphoribosyl-amino-imidazole-succino-carboxamide
synthetase (CAIR to SCAIR), and /or phosphoribosyl-amino-imidazole-carboxamide
formyltransferase (AICAR to FAICAR), and/or
phosphoribosyl-adenylo-succinate-synthase (IMP to adenylo-succinate) activities
are reduced in the case of monosomy of 5p14-5p15. This would explain the amino
acid anomalies previously described (Lejeune et al., 1990) as well as the
metabolic finding here reported.
A defect in de novo purine synthesis could account for the
characteristic failure to thrive, at birth and subsequently.
If a defect in de nova purine synthesis is indeed present in patients
with 5p- this would open a new field of research into the pathogenic mechanisms
responsible for mental retardation in patients with chromosomal anomalies. It
has been shown that in the brain, the predominant metabolic pathway for
hypoxanthine, which is derived from inosine, is towards purine nucleotides and
not towards purines such as uric acid, which cannot be salvaged by
phosphoribosyl-transferases (Spector, 1988 ; Wong et al., 1970). The importance
of purine nucleotides as neuramediators and neuro-modulators is now well
established. Any alteration in purine metabolism could therefore be responsible
for modification in brain development and function.
Our findings thus open new and exciting research perspectives. We
suggest that genes coding far enzymes intervening in the final stages of de
novo purine synthesis should be sought for on the terminal portion of the short
arm of chromosome 5. Were this indeed the case one could perhaps envision that
early substitutive therapy might be of benefit to these children.
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