Metabolic anomalies in cri du chat syndrome (5p-) lymphocytes and de novo purine synthesis

Marie A. PEETERS, Marie-Odile RETHORÉ, Luisa ARIS, A. MEGARBANE, F. CATTANEO, J. LEJEUNE

PEETERS Marie A., RETHORÉ Marie-Odile, ARIS Luisa, MEGARBANE A., CATTANEO F., LEJEUNE J. - Metabolic anomalies in cri du chat syndrome (5p-) lymphocytes and de novo purine synthesis. Ann Genet, 1991, 34, n° 3-4, 219-225.


Résumé :

SUMMARY: Having previously demonstrated that patients with cri du chat, 5p- syndrome, have a highly significant excess of the plasmatic and urinary relative amount of asparagine and aspartate, the authors tested the hypothesis according to which this excess could be in relation with a defect of purine metabolism. Using a previously reported in vitro assay, they found a paradoxal increase in the mitotic index in the presence of L-alanosine in lymphocyte cultures of patients with 5p- who were on no medication. They also observed particularly severe toxicity to HAT medium. This response, apparently characteristic for 5p- syndrome, was highly significant when compared to the one observed in samples of normal controls, of patients with mental retardation of various etiologies, patients with Down syndrome or with Xqfra syndrome. When patients with cri du chat syndrome received inosine with folinic acid, an inversion of their response to alanosine was observed as well as the normalization of their response to HAT medium. These findings suggest that deletion of 5p 14-5p15 leads to some impairment of de novo purine synthesis. the implications of these findings are discussed.

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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.
NameSexAgeKaryotypeIQTreatmentALANHAT
1HDF546,XX,del(5)(qter p14.2:)38none83 %- 97 %
2BAM 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 %
7CCF 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 thrapy4 % - 98 %
11 SCH F 30 46,XX,del(5)(qter p33.3:) 30 none6 %
12 CM F 6 46,XX,del(5)(qter p?:) 45 none 6% - 85 %
13Same as 5 folinate + inosine - 36 % - 74
14Same as 10 folinate + inosine- 17 % - 52 %
15Same 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
InosineAlanosHATPutrecine
5p- not receiving inosine n121276
m10,920,6- 8820,8
s21,722,512,813,7
Total samplen295246166135
m2,30,9- 663,4
s23,424,225,721,9
t1,22,8**2,2*1,9
Normal controlsn100764019
m2,5- 0,8- 631,3
s22,521,526,916,9
t1,23,2***2,4*2,5*
Mental retardationn76626458
m2.81.1- 696.8
s24.124.624.624.1
t1.12.5**2*1.4
Trisomy 21n59502927
m0,5- 1,4- 59-3,3
s2725.422.720.4
t1.22.7**3.2***2.7**
Xqfra syndromen24261416
m1.23.4- 63- 2.4
s18.326.52419.5
* 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
InosineAlanosHATPutrecine
5p- not receiving inosine n4443
m-6-24-686
s7.49.29.116.3
Total samplen295246166135
m2,30,9- 663,4
s23,424,225,721,9
t0.72*0.20.2
Normal controlsn100764019
m2,5- 0,8- 631,3
s22,521,526,916,9
t0.82.1*0.40.4
Mental retardationn76626458
m2.81.1- 696.8
s24.124.624.624.1
t0.72*0.10.1
Trisomy 21n59502927
m0,5- 1,4- 59-3,3
s2725.422.720.4
t0.51.70.80.7
Xqfra syndromen24261416
m1.23.4- 63- 2.4
s0.82*0.40.7
* 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
InosineAlanosAzasAdénosineHATPutrecine
5p- receiving inosine + folinate n444442
m-6-242-3-6810
s7.49.212.57.69.15.6
5p- receiving no inosinen1212101176
m10.920.6110.9-8820.9
s21.722.51826.212.813.7
t1.53.6***0.10.92.5*1
5p- receiving inosine + folinate or folinate onlyn666662
m-1.2-14.3-0.213.3-731.7
s10.418.312.718.814.714
5p- receiving no treatmentn10108956
m11.323.81.813.5-89.220.8
s252420.429.61314.9
t1.23.3***0.201.91.6
* 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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