Current research on therapeutic possibilities in trisomy 21

Jérôme Lejeune

Brighton, 11 avril 1986


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Among all the diseases affecting human intelligence, trisomy 21 is the most frequent one and, also, the most obvious. Over the personnel characteristics of the child, the disease super imposes a kind of mask producing an unmistakable "air de famille". This clinical evidence allowed LANGDON-DOWN, after SEGUIN, to isolate the condition : hence the current name of Down's Syndrome.

With their upward slanding eyelids, their little nose in a round face, their incompletly chiselled features, Downs' patients look more children than usual. Every baby has short hands with short fingers; but theirs are shorter. All their anatomy is rounded of with no asperity nor stiffness. Their ligaments and their muscles have also a suppleness producing a tender langor in them posture.

This general softness extend even to their character : cheerfull and affectionate they have special charm, easier to cherish than to describe.

That is not to say that Down's Syndrome is a desirable condition. It is an implacable disease depriving the children from the most precious quality afforded by our genetic patrimony, the full power of rational thinking.

This combination of a tragic chromosomal error with a really attracting nature reveals in a glimpse what all medicine is about : to fight against the disease and to love the disabled.

Sure enough it is now well establised that an extra chromosome 21 is the cause of Down's Syndrome. But how this super-numerary piece of genetic information produces its deleterious effect:

- how the intellectual damage is resulting from it,

- how it could possibly be compensated or repaired,

All this remains to be discovered.

Hence, instead of an historical review, I'll take the liberty of presenting you briefly the state of affairs, and discussing the various ways open to research.

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I - Types and counter-types

Normally, at the moment of conception, every child recieves two exemplars of each chromosomes : one from father, one from mother. If by misfortune one of the reproductive cells carries two elements, the child becomes trisomic ; that is he has three exemplars of chromosome 21.

Sometimes the contrary occurs, a piece of chromosome is lacking and the child is monosomic, having received only one exemplar of this segment.

If we compare the two conditions, a stricking opposition appears: the excess modify the morphology in one way and the deficit on the contrary way.

For example, trisomy 21 produces small ears with a small nose and partial monosomy 21 induces large ears with a protruding nose.

This opposition in "Type and Counter-type" is observed for all the chromosomal aberrations for which these reciprocal syndromes are known.(1). But not matter the chromosome involved, be it the 21, the 18, the 13 or the 5 or any other, when it comes to intelligence both the excess or the deficit of genetic material invariably produce the same deleterious effet: mental deficiency.

In order of resolving this paradoxe, we must remember that the genes, carried by the chromosomes, dictate the production of enzymes. Enzymes are the machine - tools of the cell, manufacturing all the biochemical products. Hence the more genes there are, the more enzymes are produced and the fastest the chemical stuff is produced or destroyed.

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The symphony of intelligence

The message of life can be compared to a symphony : each musician (the genes) reads its score and follows the tempo of the conductor.

During a solo, a too-quick musician (in case of trisomy) could transform an "andante" in a "prestissimo" : the ears will be too small and the fingers too short. conversly, a slow musician (in case of monosomy) could change an "allegretto" in a "largo" : the ear will be chiseled and the fingers too slender. In both ease, because the musician played a solo, he modified a trait but did not spoiled the whole symphony.

On the contrary, when the full orchestra is concerting, all the musician playing in a "tutti", it does not matter wether the faulty musician accelerates or slows down; the result will be cacophonic, even if he reads correctly his music!

Detecting the discarding musician is not an easy task especially when a whole chromosome is involved like in Down's syndrome. In simple genic diseases like in phenylketonuria for example, only one biochemical reaction is blocked and the failure is obvious... once discovered. But there may be some thousand genes in chromosome 21.

Surely, most of them do not produce arm when in triplicate, because if each of them was discarding, the trisomic children would not survive at all. Thus only few of the accelerated reactions are dangerous. But how will we discover the culprits among so many innocents!

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Chromosomal-regulation

This detective story could be avoided if we knew how to silence a peculiar chromosome without disturbing the others. If the extra musician was turned off, the symphony would be restored.

Let us suppose a competent repair-man has recieved from the factory a four cylinder engine equipped by mistake with flue spark-plugs. He would certainly notice that the engine does not run smoothly. An expert would not discard this motor. He would cleverly disconnect the extra plug and thus bring the rythm to normal.

Alas, we are not yet as skilled than this car repair man! But nature is that shrewed. She knows how to silence one of the X chromosomes in feminine cells, so that the woman with her two X chromosomes is not so much superior to the man who has only one X and a tiny Y!

We still ignore how this turning off is achieved. Il we could master that trick, and if we could apply it to the extra chromosome 21, we could redress the destiny of Down's Syndrome affected children, even without knowing what kind of biochemical reaction was modified by the discording musician!

Pending such a tour de force, (and nobody knows wether it could at all be feasible some day) we can try to decipher the genie content of chromosome 21.

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Genes of the 21

The painfull task of unravelling one by one the genes of chromosome 21 is less brilliant indeed, but it has the great advantage of being already on the way. Two methods are available:

- the molecular biologists split the long ribbon of the DNA contained in chromosome 21 and, letter by letter, decipher the message encoded in each piece.

- on the other hand the biochemists carefully analyses all the chemical reactions they can, in order to see if some of them are running faster in trisomic 21 cells.

We can localize the genes by analysis of the effect of small duplications or deletions, and any improvement an microscopic techniques is very much worth of ours efforts.

These approachs complement each other and engender a healtly scientific competition between the research teams.

For the moment six genes are known I and I apologize for being too technical about it: chemical language is very simple indeed, with the slight difficulty that it cannot be translated in every days words!

One, the superoxyde dismutase (S.O.D.I), plays a special role in the metabolism of oxygen (2). So to speak, it regulates the carburation. Its over activity possibly deteriorates some cell constituants, inducing the too rapid ageing of Down's Syndrome patients. They also become more frequently deficients and some of them are affected by a redoutable complication looking very much like the ALZHEIMER disease.

- Another, the phosphofructokinase (3) controles the sugar met part of the fatty acids production. Trisomics 21 are prone to diabetes and are ften overweighted.

- Two twin-genes, the glicineamide phosphoribosyl synthase (4) and the ammoimidazole phosphoribosyl synthase deal (5) with the production of purines, the building blocks of DNA. Trisomics spill over these purines as demonstrated by an increased excretion of uric acid.

- The gene responsible of the protein reactive to interferon (6) works also too fast. The trisomics are prone to various infections.

- The last one, discovered last year, is the cystathionine-ß-synthase (7) which transform homocysteine into cystathionine, a very important compound. A rabbit has less cystathionine in its brain than a dog; a chimpanzee has more than a dog, and a man more than a chimpanzee...

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The monocarbon hypothesis

To suggest some strategy for efficiently continuing the attack, we need a theoretical framework to put in order these miscellaneous informations and to conduct the next assaults.

Could it be that a peculiar chemical mechanism would be the most frequently affected in mental retardation? Few years ago I proposed that the metabolism of monocarbons could be this sensitive target (8).

In order to build the eleven thousand millions of neurons in our brain, to insulate its enormous wiring (as long as from Paris to Tokyo ) and to chisel the security keys who open and close the eleven millions of millions of connections between our neurons the brain makes an enormous consumption of monocarbons. These bits of molecules, containing only one atom of carbon (hence their name) are the smallest building stone of our nervous system, but the most used one.

Thus if the supply of monocarbons was insufficient, if its transportation by folic acid and B12 vitamine was not efficient, if its utilisation by transmethylases was not appropriate, a severe disturbance of the functionning of the brain would ensue. All that we know about the mentally deleterious effect of these impairements is in accordance with this oversimplified hypothesis.

If some very precious stuff was burned too fast by trisomic 21 children, this wastage could possibly deplete their organism of a very necessary compound.

When over-producing purines, as previously mentioned, trisomic 21 cells waste quite a lot of those precious monocarbons.

In this respect the newly discovered acceleration of the cysthionine-ß-synthase could even be of a greater interest.

In a different type of mental retardation, the homocystinuria, this very reaction is totally blacked ; exactly the contrary of the acceleration in trisomy 21. Curiously homocystinuric children are tall, slender, with long tappered fingers, just the opposite of the short stature and the short fingers of trisomics 21. Even the homocystinurics have extra creases on their fingers in contrast to the lack of some flexion creases in trisomy 21.

The type and counter-type we started with in our discussion is very obvious here (9).

And this comparison is also in accordance with the monocarbon's hypothesis. Homocysteïne, if not transformed into cystathionine, recieves a monocarbon carried by folic acid and B12 and becomes later S-adenosyl methionine ;this is the transport form of monocarbons. The next step is the construction of neurons, of insulating substances and of security keys of the nervous connections! Taken together the wastage of purine and the excessive destruction of homocysteine could produce a borderline but chronic shortage of monocarbons in trisomy 21.

On the contrary, the accumulation of homocysteine (in the blockade disease) produce S-adenosyl-homocysteine which entirely blocks the transmethylase system ; thus the blockade is just as deleterious as the acceleration

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Research on monocarbon's metabolism in trisomy 21

This hypothesis of a disturbance of monocarbon's metabolism in trisomy 21 was put forward some years ago (LEJEUNE, 1979) but no experimental protocol was avaible at that time.

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A - Oxydized monocarbons and the synthesis of purines

a) State of the art in 1984

It is known that 21 trisomic persons produce and excrete more uric acid 10 than normally (10). This excess of purine synthesis could be due to the excess of activity of phosphoribosyl-glycinamide-synthetase and of phosphoribosyl-aminoimidazole-synthetase. Both these enzymes are on chromosome 21.

This oversynthesis of purine would demand excess production of phosphoribosyl, which is produced by the shunt of hexose monophosphate ; this pathway is accelerated in trisomic 21 children.

At the end of the process, for each molecule of uric acid spilled over, two monocarbons would be lost, for the synthesis of a purine requires one formyl carried by 10-formyl-tetrahydrofolate and another one carried by 5-10-methenyltetrahydrofolate.

Hence a chronic depletion of monocarbons could ensue.

This patways seemed so much the more important that PEETERS et al.1985 (11) found that trisomic 21 children when affected by leukaemia had an over sensitivity to methotrexate ; this substance was twice as much toxic for them than for normal children affected by the same type of leukaemia.

b) Demonstration of sensitivity in vitro of trisomic 21 lymphocytes to methotrexate 1986. LEJEUNE et coll. (1986)(12)

Effects of methotrexate.

In dividing cells the synthesis of thymidine is very important. It requires the transfer of a monocarbone carried by 5-10 methylene tetrahydrofolate to deoxy-uracile-monophosphate in order to produce thymidine monaphosphate. During this reaction the tetrahydrofolate is oxydized into dihydrotetrahydrofolate. This inactive compound must be reduced again to tetrahydrofolate by dihydrofolate reductase. This last enzyme is specificelly blocked by methotrexate. This methotrexate blocks the transfer of oxydized monocarbons and blocks also the cellular division.

Hundred fifty cultures of lymphocytes were made from six trisomic 21 children and from six normal persons. The effect of four different dosage of methotrexate was studied by mesurement of the mitotic index of the cultures (i.e. the member of dividing cells aver the total number of cells).

The doses studied were 0,6 x 10-8M 1,2, 2.4, 4.8 x 10-8M.

We could thus demonstrate:

a) The mitotic index is diminished proportionnally to the square of the dose of methotrexate.b) The sensitivity of trisomic 21 lymphocytes is twice that of normal lymphocytes:

a given dose produced twice as much damage in trisomic 21 cells than in normal ones.

These two effects highly significant, are the first experimental proof that a disturbance of monocarbon's metabolism really exist in trisomy 21.

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B - Reduced monocarbons and the transmethylations.

Reduced monocarbons (methyl radicals) are used in the building of the nervous system, in the production and secondary inactivation of the chemical mediators and in all the process of methylation (production of the insulating substances like myelin and regulation of specific sites of the DNA or of the RNA).

1) State of the art in 1985

An anomaly of the homocystein's metabolisms was suspected ten years ago (9). In homocystinuria, a mental deficiency due to a block of the enzyme cystathionine-ß-synthase, the patients exhibit the countertype of trisomy 21 (they are tall, slender, with extra creases on the fingers). In 1984 SKOVBY,(7), a former student of the Institut de Progenese, demonstrated in U.S.A. that the gene of cystathionine-ß-synthase is indeed on the chromosome 21. Recently CHADEFAUX et al. 1985 (13) and JEROME et al. 1985 (14).

demonstrated that the enzyme is 1.5 time more active in trisomic 21 cells than in normal cells. They demonstrated also that the amount of newly formed methionine (from the 5-methyl-tetrahydrofolate) is less in trisomic 21 cells than in normal cells. This can be explained if, due to the acceleration of cystathionine-ß-synthase, less homocysteine is available for remethylation.

It thus seems likely that beside the trouble of oxydized monocarbones (demonstrated by the methotrexate effect), there could exist also a deficit of reduced monocarbons in trisomy 21. This hypothesis would fit with the relative cholinergic deficiency observed in trisomy 21 and with the relative deficiency of methylation of nicotinamide observed long ago by GERSHOFF, 1958 (15).

2) Work in progress

In order to investigate this open possibility, a protocol similar to the one utilized for testing methotrexate is now developped. Some new culture medium will be especially produced for this experiment. From the very preliminary data available to days it is obvious that the level of methionine in the medium affects the mitotic index ; the investigation is in progress.

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II - Clinical investigation

From the available experimental data, two ways of palliative medication can be already envisaged.

1) Increase the production of oxydized monocarbons by folic acid medication or by addition of specific precursors to the regimen.

2) Increase the production of reduced monocarbons by administration of B12 and B6 vitamines, as well as methionine or homocysteine or other derivative of this particular pathway.

All these assay will utilise, at physiological doses, substances normally present in the food and in the healtly organism.

Possible changes in the speed of mental acquisitions (IQ) will be controlled by psychometric tests. The service in the Hopital des Enfants Malades, is fully equipped and staffed for this research.

Although some of these investigations are already on the way with some, preliminary results not too discouraging, the matter is so important and touchy that no comment can be included in the present report.

It must be very precisely stressed that this general model is for the moment strictly speculative. Even if the reasoning was sound, it would remain to be seen wether the correction of such a trouble, if at all possible, could alleviate the mental deficiency. A specific treatment is yet to be discovered.

No specialist can for-see the length of the road to be covered, until reaching such an achievement, but the future of research sounds very much like a wellknown song : "we shall overcome some day".


Fig.1


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References

(1) LEJEUNE J. - Types et contre typres. Journées parisiennes de Pédiatrie. 75-83. Ed. Med. Flammarion 1966.

(2) SICHITIV S., SINET M.M., LEJEUNE J., FREZAL J. - Surdosage de la forme dimérique de l'indophénoloxydase dans la trisomie 21, secondaire au surdosage génique. Humagenetik. 1974, 23, 65.

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

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

(5) PATTERSON D., GRAW S., 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. 1981, 78, 405-409.

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

(7) SKOVBY R., KRASSIKOFF N., FRANCKE V. - Assignment of the gene for cystathionine-ß-synthase to human chromosome 21 in somatic cell hybrids. Hum. Genet. 1984, 65, 291-294.

(8) LEJEUNE J. - Le metabolisme des monocarbones et la débilité de l'intelligence. in : La débilité mentale p. 3-18. Masson Editeur 1983.

(9) LEJEUNE J. - Réflexions sur la débilité de l'intelligence des enfants trisomiques 21. Pont. Acad. Sc. Rome, 1975, 9, 1-12, commentarr III.

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

(11) PESTERS M., POON A.L., ZIPURSKY A., OLIVE D. - Mongolisme et leucémie : toxicité accrue au méthotrexate. Compte rendu du Congrès National d'Hématologie et de Transfusion Sanguine. Bordeaux, 1985, p. 71.

(12) LEJEUNE J., RETHORÉ M.O., BLOIS M.C. de, MAUNOURY-BUROLLA C. MIR M., NICOLLE L. BOROWY F., BORGHI E., RECAN D. - Metabolisms des monocarbones et trisomie 21 sensibilité au méthotrexate. Ann. Genet. 1986, 29, n° 1, 16-19.

(13) CHADEFAUX B., RETHORÉ M.O., CEBALLOS I., ALLARD D. - Effet de dosage génique et consequences métaboliques de la trisomie 21. Académie Nationale de Médecine, seance du 3 décembre 1985.

(14) JEROME H., CHADEFAUX B., CEBALLOS I., ALLARD D. - Effet de dosage génique et consequences métaboliques de la trisomie 21. Académie Nationale de Médecine, seance du 3 décembre 1985.

(15) GERSHOFF S.N., HEGSTED D.M., TRULSON M.F. - Metabolic studies of mongoloids. Am. J. Clin. Nutr, 1958, 6, 526-530.