Therapeutic reseach in trisomy 21 a heuristic model

LEJEUNE Jérôme, PRIEUR Marguerite, RETHORÉ Marie Odile, AURIAS Alain et RAOUL Odile.

Congrès de mexico, mars 1983


Sommaire

Down's Syndrome, the most frequent cause of mental retardation is also the most studied. Nearly a quarter of a century after the discovery of the chromosomal basis of the disease (1) the crop of scientific results is impressive. To summarize this enormous bulk of knowledge is the very purpose of this whole Congress. Various genes have been located on the 21 chromosome and it is definitely established that the patients owe their disease to an excess of genetic material, otherwise normal. Genic dosage effect, hypothesised long ago (2) is now an experimental fact.

In contrast with this scientific exuberance the therapeutic side of the research is still in infancy.

Alleged "cures" and so-called "treatments" are published nearly every year, but no claim has yet withstood scientific scrutiny. This objective judgement apply to the medication and not to the rehabilitation and training techniques, which are still the best and only aid we can presently offer to the affected children.

In view of this terrible lag in therapeutic research, it seems worth investigating how a heuristic program could be figured out.

In order to do so, three points must be discussed :

1) Does the actual knowledge point toward a particular biochemical trouble : a general framework of research?

2) Is it possible to build a model of the spontaneous evolution of the mental performance of trisomic 21 children so that an eventual therapeutic effect could be tested?

3) How could clinical trials be divised according to 1 and 2?

Haut

I . A general framework for research.

As already proposed 4 years ago (3), a general discussion of the metabolic blocks resulting from gene mutations and producing mental deficiency, point to the metabolism of monocarbons as a very sensitive mechanism. Already fragile-X syndrome, another chromosomal disease related to mental deficiency, can be related to this monocarbon metabolism (4).

For trisomy 21, the relevance of this metabolism is much more difficult to assess. Nevertheless, some of the available data seems compatible with this general hypothesis.

Haut

A) Genes on the 21 and the phenotype of the trisomy 21 syndrome.

Five genes have been assigned to chromosome 21, namely : superoxyde dismutase-1 (soluble), phosphoribosyl glycinamide synthetase, phosphoribosyl aminoimidazole synthetase, antiviral protein (interferon receptor) and phosphofructokinase (liver-type)

a) Superoxyde dismutase 1

Superoxyde dismutase-1 was the first example in man of a gene dosage effect resulting from a trisomy (5). This enzyme is 1"5 times more active in trisomic 21 children than in normal ones. Various translocations have shown that its locus is around the zone q22.1 of the long arm of 21 (6).

This excess of activity is expected in trisomics, because they have three chromosomes 21 instead of the normal two. Hence if the quantity of enzyme produced is proportional, to the number of genes, the activity ratio must be 3 to 2. i.e a/1.5 fold ncrease.

The effect on the phenotype is difficult to predict. S.O.D.1. transmutes the superoxyde ion O2- to H2O2 by a complex mechanism. The superoxyde ion O2- is extremly reactive and is often considered as a toxic : it can oxydize the fatty acids of the membranes and damage them gravely. Such a mechanism has been postulated by some theories about ageing process. In the organism the superoxyde ion is nevertheless used in some particularly important reactions : indoleamine dioxygenase for example.

The hydrogen peroxyde, produced by superoxyde dismutase-1 must be disposed of, for H2O2 is also a very active oxydant. The reaction H2O2 ? H2O is controlled by glutathione peroxydase (GPX).

The GPX activity is augmented in trisomy 21 (7), although its gene is located on chromosome 3. This excess of activity, obviously a regulation effect, is particularly interesting for it shows a significant correlation with the I.Q. of the patients : the greater, the I.Q. of a child the higher the GPX activity of his red-cells. It could be hypothesised that the increase of GPX activity protects the tissues against the excess of H2O2 produced by superoxyde dismutase.

b) Phosphofructokinase

This enzyme of the glycolytic pathway is also 1,5 times more active in trisomic 21 than in normals (8). This enzyme transforms the fructose-6-phosphate into the fructose-1-6-diphosphate. This acceleration of one of the first steps of glycolysis could be related to the well reported (9) instability of the glycemia. Very often (10) (11) trisomics have been described as "pre diabetics" although true diabetes does no seem more frequent than in the general population.

A secondary effect of this accelerated transformation could be related to the fact that fructose-1-6- diphosphate itself increases the activity of another enzyme, the acetyl-CoA carboxylase biotine dependant ; acetyl-CoA is carboxylated to produce malonyl-CoA which is the starting point of the synthesis of fatty acids. It is an open possibility that stimulation of this step could have some relation to the well known tendancy to obesity, so frequent in Down's Syndrome.

c) Phosphoribosyl glycinamide synthetase and phosphoribosyl aminoimidazole synthetase.

These two enzymes belong to successive steps producing the purine bases in vivo.

The increase of their activity (12) (13) could lead to an excessive production of these nucleotides, indispensable for the building of nucleic acids molecules (DNA and RNA) .

If it was so, such an over production should be accompanied by two biochemical symptoms : 1° the turnover of phosphoribosyl pyrophosphate should be increased. 2° the excess of bases should be disposed of via uric acid. And both phenomenous are observed in trisomic 21.

The production of phosphoribosyl pyrophosphate is achieved via the shunt of the hexose monophosphates and this particular pathway is faster in trisomics (14), although the amount of the respective enzymes is not increased appreciably.

The elimination of uric acid is augmented in Down's Syndrome as it was observed long ago. Even in the plasma, there is a significant excess (15) not only of uric acid but also of xanthine and hypoxanthine bases.

All together these various data point toward an over production of the purine bases fitting with the gene dosage effect.

d) Interferon reactive protein

It is now established that trisomic 21 lymphocytes have more sites responsive to interferon than normal (16) (17). It could be surmised that this could affect the immunologic reactions and induce a compensatory rise in gamma globulin.

These relationships between the five genes known to be on the 21 chromosome and the phenotype of the patients are plausible. However it remains to be seen if these troubles could play some role in the monocarbons metabolism.

Haut

B) A possible deficit in the monocarbons metabolism.

Keeping in mind the long synthetic pathway of purines one can see (fig. 8) that monocarbons are required at two different steps (one carried by the 10-formyl tetrahydrofolate and the other by the 5-10 methenyl tetrahydrofolate). Precisely two monocarbon moieties are lost for each excreted molecule of uric acid. It follows that ah. unnecessary synthesis of purine, secondarily excreted as uric acid, would require an extra amount of monocarbons which would be at the end, spilled over.

Monocarbons are probably in limited supply (18); thus this overuse could constitute a severe depletion in the organism.

Apparently, superoxyde dismutase hyperactivity has nothing to do with monocarbons. Curiously a series of experiments (19) carried out on the fragile-X Syndrome, have demonstrated an interesting correlation. If the purified enzyme is added to the culture medium for a sufficiently long time (72h or 48h before harvesting) it diminishes remarkably the frequency of the gap seen at the end of the long arm of the X chromosome (site Xq28). From this experiment it can be inferred that the level of superoxyde ions has some effect upon the monocarbon metabolism which is definitely very sensitive in this disease.

Before accepting the hypothesis that a trouble of monocarbon metalolis could result from the gene dosage effects already qu ted, other symptoms of trisomy 21 must be analysed.

a) Hypocholinergy in trisomy 21

As demonstrated long ago, trisomic 21 children are hypersen-sitive to atropine (20). A systematic study of the pharmacologic reactivity of their iris showed hypersensitivity to cholinomimetic and adrenolytic compounds ; a low production of cholinergic mediators seems the simplest explanation (21). In this respect, it must be remembered that the production of each molecule of acetylcholine, requires three monocarbons in order to trimethylate the ethanolamine.

b) Pseudo Alzheimer complication

Trisomic 21 adults are very prone to precocious senility. More dramatically, the frequency of Alzheimer-like syndrome is greatly increased (22). In both diseases the ultra structural anomaly of the entangled neuro-filaments is typical. Cholinergic troubles are well know in Alzheimer disease : deficiency of acetylcholine esterase and of choline acetyltransferase, with disorder of cortical cholinergic innervation (23).

c) Pseudo Lesch-Nyhan complication

In rare case of severly retarded trisomic 21 children, an agressive and even auto-agressive behaviour is sometimes observed. This behaviour is reminiscent of the Lesch-Nyhan disease, a hyper-uricemic condition due to a deficiency of the hypoxanthine-guanine-phosphoribosyl-transferase. These patients cannot recuperate free guanine (for recycling it in the ribonucleotide pathway) and thus suffer of a very severe loss of purines, excreted as uric acid. This imply a very severe demand upon the monocarbon metabolism.

It remains to investigate on a large number of pseudo Lesch-Nyhan trisomic 21 children, whether they have especially high blood levels of uric acid and especially high excretion.

d) A possible deficit of methylation

These three clinical findings, hypocholinergy, tendancy to Alzheimer disease and pseudo Lesh-Nyhan behaviour do not demonstrate a monocarbon trouble but are not in desagreement with the hypothesis.

Remarkably, in 1958 GERSHOFF and Al., (24) came to the same conclusion. Studying the urinary excretion of trisomic 21 children challenged with high doses of niacin, they discovered that the patients excreted less creatinine and less N-methyl-nicotinamide than controls (nicotinamide is excreted after methylation) . GERSHOFF concluded then that, possibly, trisomic 21 children had a diminished methylation power. A potentially very important conclusion which, curiously, remained quite unnoticed for a quarter of a century!

To sum up this rapid review we can simply state that the general hypothesis of monocarbon disturbancies in mental retarda-tion, already strongly suspected in the fragile-X syndrome, could be applied as an heuristic model to Down's syndrome investigations.

Let us now turn to the second question : how could we devise a test in order of demonstrating a possible relationship between monocarbon metabolism and mental deficiency in Down's Syndrome? Obviously the first step would be to quantify the mental evolution of the children in order to see secondarily if any correction of the monocarbon metabolism could ameliorate their development.

Haut

II. A quantitative approach of the mental deficiency in down's syndrome

Haut

A) A follow up during fourteen years

In order to follow the mental evolution of Down's syndrome children, we have selected the case history of 183 children (94 boys and 89 girls) followed at the Institut de Progenèse at the specialised consultation in the Hopital des Enfants Malades à Paris. Most of them have been examined and tested every year or twice a year and followed from one year to 14 years of age ; the chosen cohorte, was only selected on the basis of the date of birth, between January 1968 and January 1973.

Haut

B) Psychometric examinations

No one psychometric test is available for the entire follow-up from one year to 14 years of age. Hence three different tests have been used according to the actual performances of the patients. From 10 months of age up to three years, all the patients are subjected to the "test de developpement de la premiere enfance de Brunet-Lezine". - 143 children have been tested at least once with this test.

After 3 years, the best developed patients satisfy all the items of the Brunet-Lezine test and are then subjected to the "test sans parole de Borel-Maisonny" ; the less rapid children continue with the Brunet-Lezine.

At around 4-5 years of age, practically all the children are tested with the Borel Maisonny non-verbal test.

Later, the development of the language is so important that non-verbal tests become irrelevant. Then the classical Binet-Simon tests is relied upon. At 8 years, 75 % of the patients are tested with the Binet-Simon and 90 % at 11 years.

Haut

C) Difficulty of this follow-up

The choice of these three tests is open to criticism. Other more refined protocols could have been used as well. This battery has the advantage of simplicity and our team of dedicated psychologists are well accustomed to them.

Another imperfection, (and no training of the psychologist can overcome it) stems from the fact that the estimation of the performances (the "mental age") is not exactly identical with two tests at the zone overlapping. As it can be seen in the figures 1 to 7, this unescapable difficulty do not blurr too much the general picture.

The main interest of this compound statistics is that every child can be followed individually and that groups of children of comparable performances at a given age can be followed in their or future (or previous) development.

More generally one can also calculate at each chronological age, the mean mental age and estimate the standard deviation. If this process is completed by a "smoothing of the curves" one can establish the figure 1 in which the mean mental age is represented, with the curves corresponding to one and two standard deviations.

It must be emphasized that the distribution of these mental age, for each chronological age is not perfectly qaussian, 93 % of the children are within the limits (m + 2s) and (m - 2s), But if the extrems are considered, 2 % are better than (m + 2s) and 5 % are below (m - 2s). The distribution is slightly asymetrical. This is due mainly to cohort of children who make very slow progress, instead of following the general trend.

A manner of visualizing the individual evolution is to choose the age class 11 to 12 years. For this class the population can be seperated according to quantiles.

The figures 2, 3, 4, 5, 6, 7 exemplify very clearly the homogeneity of the pathway if it is reconstructed for all the children achieving a given mental efficiency at 11-12 years period.

It follows that for each child the predictive value of his actual achievment is quite good. The cohort reaching the upper level, cross the barrier of 5 years of mental age much earlier than the cohort achieving just 5 years at the same age. The width of the path for a cohort is generally smaller than 2 of the general population. It must be stressed at this point of the analysis that all the data have been split according to sexe ; there is no difference between boys and girls for all the trends analysed.

Haut

3) The "relative progress"

As shown by the general distribution the standard deviation is not constant with ageing : the s much greater at 12 years of age than at 1 year for example.

To normalize the estimation of the "progress" during a given period (six months in most of the experiments described later) a new parameter can be chosen : the "relative progress".

If a child has attained a given mental age at a given chronological age,(dark circle)at the left of the figure 1, it is expected that, six months later, his performance (open circle) would lie on an imaginary line, pseudo parallele to the lines of the standard deviation of the general population. If the observed point (second dark circle) is higher than this predicted trajectory the difference, the observed gain (a), is measured. In order to standardize this value (expressed in months of mental age), it is divided by the expected standard deviation (b).

Thus, the "progress" or the "regression" are expressed as a percentage of the standard deviation : the "relative progress".

It follows from this procedure that the mathematical expectation of the "relative progress" is zero, (if the tables are representative for all the children). Moreover, if this procedure is repeated all along the evolution of a child it allows to control the regularity of its trajectory.

For prediction purpose it must be checked, wether the observed "relative progress" is nil (as the mathematic expectation would predict) or whether it is significantly lower or higher.

This has been checked systematically on 529 six months changes and the observed "relative progress" was as a mean + 2,10 ± 46,14. That is two percent of the standard deviation, with a sof half a standard deviation. Hence the observed value is as close to zero as it could be expected. Each child follows its own personal paths, with ups and downs y but his whole trajectory is inscribed in 95 % of the cases within borders of +1 sand -1 sof the general distribution.

This predictive value of the "relative progress" is of extreme importance, because it indicates that if a sample of say 20 to 25 children submitted to a given treatment, were experiencing as a mean an increase or a decrease of greater than 1 sof the general population, this would be highly significant.

Without going into too much statistical details, it can be stressed that if the "relative progress" is tested against its own standard error, "t" Student statistics will detect the eventual significance of the change.

Having thus at hand the clinical instrument, it was possible to settle a heuristic investigation.

Haut

Ill. A heuristic analysis of mental development. According to attempts of modification of the monocarbon metabolism in trisomic 21 children.

Haut

A) heuristic clinical trials

Coming back to the central hypothesis of a deficit of monocarbon's metabolism, it becomes clear that correction could possibly be achieved by three different interventions.

1) A supply of precursors of monocarbons could increase their availability. This could be achieved by administration of any of the precursors listed in figure 8 (column of precursors).

2) Amelioration of the transportation system could also increase the amount of monocarbons available to the organism. This could be achieved by administration of the vitamines folic acid and cobalamin (and their derivatives) figure 8 (column of monocarbons).

3) An alleviation of the demand of monocarbons could be obtained if final products were supplied. This could be achieved by administration of extra amounts of purines or of carnitine figure 8 (column of synthesis).

A systematic check of these possibilities is underway and the present stage can be summarized as follow.

Haut

B) Administration of precursors

Among the products listed in the first column, tryptophan and 5-hydroxytryptophan were excluded. They have already been tested by other workers using a very different rationale. Tryptophan did not gave appreciable results (25) and 5-hydroxytryptophan gave either inconclusive results at low doses (26)(27) or dangerous effect in high doses (28), a number of West Syndrom's (hypsarythmia) was provoked by this treatment. It must be stres-sed that the choice of 5-hydroxytryptophan was a seemingly favourable bet, for it has long been known (29) that trisomic 21 children have a trouble of the tryptophan metabolism : an accelaration of the kynurenin pathway. They have also a low level of serotonin, due to a defective pumping mechanism in their platelets (30) (31) (32).

Histidine has not yet been checked, nor 5-aminolevulinate because of their possible toxicity as experienced in histidinemia or in porphyrias. This caution does not preclude at all their possible interest.

Serine seemed a better choice especially because trisomic 21 children have a slight deficiency of serine in their plasma together with an excess of ethanolamine (33). Also 4-hydroxyproline was tested, considering the phenotypic similarities between trisomy 21 and iminodipeptiduria (34) (35) a rare disease in which, due to the lack of an enzyme, patients loose each day some 10 to 20 mg of hydroxyproline.

Awaiting the availability of N.N.dimethylglycine (now under experimental trial) a very much used derivative, but non natural, was tested : dimethyl amino-ethanol.

The results are summarized in table 1.

In a preliminary trial at dose of 25 mg/kg/d serine appeared promising with a "relative progress" of + 26,82 ; s= 81,95. A second test using 50 mg/kg/d looked also beneficial with a "relative progress" of 24,93 ; s= 33,79 (table II).

This prompted us to start a full double blind test in which serine at 100 mg/kg/d, was tested, together with proline 100 mg/kg/d and hydroxyproline 50 mg/kg/d, carefully randomized with placebo on 93 children.

All these trials were made on children more than 4 years old and less than 9 years of age. This interval is possibly not the most sensitive one, but it was selected in view of two facts : first the curves of mental development are quite linear in this interval. Second the parents could easily detect any unforeseen deleterious effect. This would not be the case in newborn (28). Indeed if the medication is really good, the children would react better, the younger they are. But "primum non nocere" is an absolute prerequisite.

All the trials are performed with the fully informed consent of the parents. They understand that these assays are no definite treatment but systematic research of an eventual sensitivity of trisomic 21 children.

The fully randomized trial gave perfectly null results as seen in the table II. This is a warning, if at all necessary, against premature conclusion of uncompletly controled trials.

To sum up the available results, we have now indication that neither serine nor dimethyl amino ethanol can be taken as efficient , although a possible dose/effect could still be discussed. All the same, administration of final products, purines or methionine or relevant vitamines, folic acid (*) and B 12 (see table I) cannot be detected as beneficial with this procedure.

Haut

C) Toward the next step

Before rejecting the general hypothesis we started with, numerous investigations are still to be conducted. As already said, 5-amino levulinate, histidine and N.N.dimethylglycine are still to be investigated.

On the other hand, end products could be tested, and among them carnitine which is a very important metabolite. This trimethylated molecule is the carrier of fatty acid residues inside the mitochondria. It is thus indispensable for muscular foncfion and its synthesis from hydroxylysine is very demanding upon the monocarbon's metabolism. Carnitine given orally, can alleviate the neurological troubles of children affected by carnitine synthetase deficiency (36). This shows that it can be directly used from the regimen.

Its possible effect could be twofold : alleviate the demand upon the monocarbon metabolism and increase the turn-over of fatty acids.

Another intervention is still open : a direct modification of the methionine/homocysteine equilibrium. Homocysteine has not yet been administered in View of its potential toxicity in homocystinuria (37) and of its convulsing properties in animals (38).

But the interest of such an investigation is not to be over-looked. As already noted (3), homocystinuria produces quite the countertype of the phenotype of trisomy 21. Homocystinurics are tall, slim, with extra creases on fingers, in contrast with the trisomic 21 who are short, bulky and lack some flexion creases.

In this context it can be noted that homocysteic acid stimulates the growth in rats (39).

Moreover, the two diseases have in common, beside the mental retardation a peripheral vascular trouble : frequent livedo reticularis and rosy cheeks.

Another reason for checking the homocysteine pathway is the deficit in urinary taurine (by-product of cystathionine metabolism, see (fig. 8) observed in trisomy 21 (40)(41).

Although methionine and homocysteine have a very intricate regulating action on the whole monocarbon's metabolism, a cautious clinical assay does not seems out of reach.

Haut

Conclusion

This review of the present stage of research on a medical treatment of trisomy 21 is possibly not as negative as the few results obtained so far.

Sure enough, no proven therapy has yet been found, but a heuristic model can be presented and tested. The hypothesis of a trouble of the monocarbon pathway is far from demonstrated but up to now, there is no experimental fact to contradict it formally. Moreover the model is susceptible of systematic investigation for eventual disproval.

But the time factor must no be disregarded : the few inconclusive data discussed here represent six years of efforts. In this context it is a duty and a pleasure to pay homage to the marvellous dedication of the parents and to the willingness of the children in complying to very precis schedules.


Fig. 1- Mean and standard deviation of the mental age of trisomic 21 children, according to their chronological age. The example given explains the calculation of "the relative progress". See text for explanation.


Fig. 2- Path of children reaching a mental age below 3 years at 11 years of chronological age.


Fig. 3- Path of children reaching a mental age between 3 and 4 years, at 11 years of chronologic al age.


Fig. 4- Path of children reaching a mental age between 4 and 5 years, at 11 years of chronological age.


Fig. 5- Path of children reaching a mental age between 5 and 6 years, at 11 years of chronological age.


Fig. 6- Path of children reaching a mental age between 6 and 7 years, at 11 years of chronological age.


Fig. 7- Path of children reaching a mental age above 7 years, at 11 years of chronological age


Fig. 8- Monocarbon metabolism.

Table I : Open trials of products interfering with monocarbon metabolism. Trisomic 21 children from 4 to 9 years old.
Tested productMean doseNumber of children"Relative progress"Standard deviation
Controlsnothing529+2,1046,14
L-serine50 mg/k/d27+24,9333,79
L-proline10 mg/k/d.8+10,3042,06
Di-methyl5-10 mg/k/d.34+2,0343,12
amino-ethanol20-30 mg/k/d.21+17,9550,92
L-methionine50 mg/k/d.23+3,0359,0
Nucleotides10 mg/k/d.30-2,0164,0
Folic acid0,5 mg/k/d.47-1,4534,76
Table II : Double-blind trial on 93 children, trisomics 21, treated during 6 months. Age range : 5 to 8 years.
Tested productMean doseMumber of children"Relative progress"Standard deviation
Placebo100 mg/k/d.24+4,5437,03
L-serine100 mg/k/d.24-6,5450,78
L-proline100 mg/k/d.25-7,7641,24
L-4-hydroxy-proline50 mg/k/d.20-11,6036,39

Haut

Note

(*) Preliminary assays of folinic acid (5-formyl-tetrahydrofolate) in severly retarded trisomic 21 children, exhibiting psychotic symptoms or Alzheimer-like complication have given us some favourable results.

Haut

Bibliography

1 - LEJEUNE J., GAUTIER M. et TURPIN R. Les chromosomes humains en culture de tissus. C.R. Acad. Sci. Paris. 248, 602-603 (1959)

2 - LEJEUNE J. Autosomal disorders. Pediatrics 52, 326-337 (1963)

3 - LEJEUNE J. Investigations biochimiques et trisomie 21. Ann. Génét. (Paris) 22, 67-75 (1979)

4 - LEJEUNE J. Le metabolisme des monocarbones et Ie syndrome de l'X fragile. Bull. Acad. Nat. Med. 165, 1197-1206 (1981)

5 - SINET P.M., ALLARD D., LEJEUNE J., JEROME H. Augmentation d'activité de la superoxyde dismutase erythrocytaire dans la trisomie pour le chromosome 21. C.R. Acad. Sci. Paris 278, série D. 3267-3270 (1974)

6 - SINET P.M., COUTURIER J., DUTRILLAUX B. , POISSONNIER M., RAOUL O., RETHORE M.O., ALLARD D., LEJEUNE J. et JEROME H. Trisomie 21 et Superoxyde dismutase 1 (IPO-A). Exp. Cell. Res. 97, 4755- (1976)

7 - SINET P.M., MICHELSON A.M., BAZIN A., LEJEUNE J. et JEROME H. Increase of Glutathion peroxydase activity in erythrocytes from trisomy 21 subjects. Biochem. Biophys. Res. Comm. 67, 910-915 (1976)

8 - VORA S. and FRANCKE U. Assignment of the human gene for liver-type-6-phosphofructokinase isozyme (PFKL) to chromosome 21 by using somatic cell hybrids and monoclonal anti-L antibody. Proc. Natl. Acad. USA 78, 3738-3742 (1981)

9 - RUNGE G.H. Glucose tolerance in mongolism. Amer. J. Ment. Defic. 63, 822 (1959)

10 - RAITI S., LIFSCHITZ F., TRIAS E. et SIGMAN B. Down's Syndrome study of carbohydrate metabolism. Acta Endocrinologica 76, 506-512 (1974)

11 - SUTNICK A.I., LONDON W.T., GERSTLEY B.J.S., COYNE BLUMBERG B.S. et LUST-BADER E.D. Glucose tolerance in Down's Syndrome. Res. Comm. Chem.. Path. Pharm. 8, 471-480 (1974)

12 - MOORE E.E., JONES C., KAO F.T. et OATES D.C. Synteny between glycinamide ribonucleotide synthetase and superoxyde dismutase (soluble). Am. J. Hum. Genet. 29, 389-396 (1977)

13 - PATTERSON D., GRAW S. et JONES C. Demonstration, by somatic cell genetics, of coordination of genes for two enzymes of purine synthesis assigned to human chromosome 21. Proc. Natl. Acad. Sci. USA. 78, 405-409 (1981)

14 - SINET P.M., LEJEUNE J. et JEROME H. Trisomy 21 (Down's Syndrome) Glutathione Peroxydase, Hexose Monophosphate shunt and IQ. Life Sciences 24, 29-34 (1979)

15 - APPELTON M.D., HAAB W. , BURTI U. et ORSULAK P.J. Plasma urate, levels in mongolism. Am. J. ment. Def. 74, 196-199 (1970)

16 - TAN Y.H., TISCHFIELD J. et RUDDLE P.M. The linkage of genes for the human interferon induced antiviral protein and indophenol oxidase B traits to chromosome G 21. J. Exp. Med. 137, 317-330 (1973).

17 - MOGENSEN K.E., VIGNAUX F. et GRESSER I. Enhanced expression of cellular receptors for human interferon a on peripheral lymphocytes from patients with Down's Syndrome. FEBS letters, 140, 2, 285-287 (1982)

18 - MUDD S.H. et POOLE J.R. Labile methyl balances for normal humans on various dietary regimens. Metabolism 24, 721-735 (1975)

19 - LEJEUNE J. Le metabolisme des monocarbones et la debilité de l'intelligence. Congrès latino-américain de Génétique humaine. Genetica. 23-34 (1982)

20 - BERG J.M., BRANDON M.W.G. et KIRMAN B.K. Atropine in mongolism. Lancet 2, 441 (1959)

21 - LEJEUNE J., BOURDAIS M. et PRIEUR M. Sensibilité pharmacologique de l'iris des enfants trisomiques 21. Thérapie 31, 447-454 (1976)

22 - JERVIS G.A. Premature senility in Down's Syndrome. Ann. New York Acad. Sci. 171, 559-561 (1970)

23 - COYLE J.T., PRICE D.L., DELONG M.R. Alzheimer's disease : a disorder of cortical cholinergic Innervation. Science 219, 1184-1190 (1983)

24 - GERSHOFF S.N., HEGSTED D.M. et TRULSON M.F. Metabolic studies of mongoloids. Am. J. Clin. Nutr. J5, 526-530 (1958)

25 - AIRAKSINEN E.M. Tryptophan treatment of infants with Down's Syndrome. Ann. Clin. Res. 6, 33-39 (1974)

26 - CRUZ M., TORRALBA A., RODRIGUEZ HIERRO F. et MUNOZ LOPEZ F. Le traitement des enfants mongoliens avec un nouveau dérivé du 5-hydroxytryptophane. XIII Congrès International Pédiat. Wien 1971 III-107, 493-499

27 - PUESCHEL S.M., REED R.B., CRONK C.E. et GOLDSTEIN B.I. 5-hydroxy-tryptophan and pyridoxine. Their effects in young children with Down's Syndrome. Am. J. Dis. Children 134, 838-844 (1980)

28 - COLEMAN M. Infantile spasms associated with 5-hydroxytryptophan administration in patients with Down's Syndrome. Neurology (Minnéapolis) 21, 911-919 (1971)

29 - JEROME H., LEJEUNE J. et TURPIN R. Etude de l'excretion urinaire de certains métabolismes en tryptophane chez les enfants mongoliens. C.R. Acad. Sci. Paris 251, 474-476 (1960)

30 - JEROME H. et KAMOUN P. Platelet binding of Serotonine. Ann. New York Acad. Sci. 171 , 537-542 (1970)

31 - McCOY E., SEGAL K.J., BAYER S.M. et STRYNADKA K.D. Decreased ATPase and increased sodium content of platelets in Down's Syndrome. New. Engl. J. Med. 291, 950-953 (1974)

32 - McCOY et ENNS L. Sodium transport, ouabain binding and (Na+/k+) ATPase activity in Down's Syndrome platelets. Pediat. Res. 12, 685-689 (1978)

33 - SINET P.M. Contribution à l'analyse statistique des résultats de dosages d'acides aminés sanguins. Thèse de Médecine Paris (1972)

34 - GOODMAN S.I., SOEOMDNS.C.C., MUSCHENHEIM F., McINTYRE C.A., MILES B. and O'BRIEN D. A syndrome resembling lathyrism associated with iminodipeptiduria. Am. J. Med. 45, 152-159 (1968)

35 - JACKSON S.H., DENNIS A.W. and GREENBERG M. Iminodipeptiduria : a genetic defect in recycling collagen ; a method for determining prolidase in erythrocytes. C.M.A. Journal 115, 759-763 (1975)

36 - CHAPOY P.R., ANGELINI C., BROWN J., STIFF J.E., SHUG A.L. and CEDERBAUM S.D. Systemic carnitine deficiency. A treatable inherited lipid-storage disease presenting as Reye's syndrome. New. Engl. J. Med. 303, 1389-1394 (1983)

37 - GRÖBE H. Homocystinuria (Cystathionine synthase deficiency). Results of treatment in late-diagnosed patients. Eur. J. Pediatr. 135, 199-203 (1980)

38- FREED W.J., TAYLOR S.P., LUCHINS D.J., WYATT R.J. and GILLIN J.C. Production of convulsions in mice by the combination of methionine and homocysteine. Psychopharmacology 69, 275-280 (1980)

39 - CLOPATH P., SMITH V.C. and McCULLY K.S. Growth promotion by homocysteic acid. Science 192, 372-374 (1976)

40 - McCOY E.E., ANAST C.S. and NAYLO.R J.S. The excretion of oxalic acid following deoxypyridoxine and tryptophane administration in mongoloid and non mongoloid subjects. The J. Pediatrics 65, 208-214 (1967)

41 - WAINER A., KING J.S., GOODMAN H.O. and THOMAS J.J. Taurine 355 metabolism in normal and mongoloid individuals. Proc. Soc. Exp. Biol. Med. 121 , 212 (1966)