Research on pathogeny of mental retardation in trisomy 21

Jerome lejeune

Pontificia Academia Scientiarum. Working group on: "aspects of the uses of genetic engineering" Date: October 19-23, 1987. Commentarii Vol. III N° 31. 1-18


The most obvious therapy of a trisomic condition would be to silence the extra chromosome [1] so that only a diploid amount of genetic information would remain active. Indeed nature is that shrewd and inactivation of supernumerary X chromosomes is a very efficient trick. Unfortunately the basic mechanism is still unknown, although the recent use of anti-sense RNA gives already the possibility of silencing some specific sequences [2].

Pending the "tour de force" of inhibiting in every cell one chromosome 21 among the three present in Down's Syndrome affected children, this elegant way of getting rid of an undesirable genie overdose is, for the moment, out of reach. Hence the tedious and laborious comparison of the clinical data and of the DNA deciphering is, currently, the starting point of any pathogenic scheme.


The molecular biology approach

By digesting, transferring, cloning, hybridizing and blotting various stretches of DNA (or RNA) sequences, the contents of chromosome 21 can be progressively analyzed. Optimists consider that the total sequence of the 21 could possibly be unraveled in some 5 to 10 years. With the increasing automation of all the steps involved, this optimistic view could very well turn out to be a quite realistic one. But spelling out every base of the DNA sequences of chromosome 21 is not the final answer. Remains to understand the properties of the proteins coded, the functions of the enzymes and the complex activity of other stretches: activators, repressers, enhancers etc... etc. Time prophecies are quite dangerous in front of such an enormous task.


Clinical chemistry and mental retardation

Awaiting the expected results of DNA exploration, another way of investigation can be opened by the apparently very naive question: "Why are trisomic 21 children mentally retarded?" Or, in more operational terms, "How could a genie overdose hamper the functionning of their brain?".

We know for sure that the genie information carried by the extra chromosome is normal (as demonstrated by the trans-location cases). Then we are forced to look for some metabolisms in which a shift of the equilibrium between the specific speeds of biochemical reactions could be, per se, deleterious.

In 1979, a trouble of monocarbon's metabolism was presented as the possible sensitive target in mental retardation [3].

This proposal [69] did not raise discussions after it was applied to 21 trisomy in the Down's.


The monocarbon's hypothesis

A schematic drawing (fig. 1) reveals the complexity of the monocarbon's metabolism.

Fig. 1. Genetic diseases and specific biochemical abnormalities related to severe neurological and/or mental deficiencies. Legends of fig. 1: see annexe.

From the raw material like FIGLU, DOVA, etc, (see 1 to 7 columns of precursors) monocarbons are transferred to tetra-hydrofolate (8 to 11 column at left) and part of them, the oxidized forms, are directly injected in the purine synthesis (third column, 14 to 19).

At both ends, the monocarbons, at the reduced levels, are used to synthesize thymidine (12) or to remethylate homo-cysteine into methionine via 5-methyl THF and B12 (23, 24, 25).

In the middle of the scheme, the enzymatic machinery handling the tetrahydrofolate derivatives, in close connexion with the one related to biopterin metabolism looks like a maze or the blueprint of a quite intricate plumbing system.

For example 5-10 methylene THF reductase (20), normally reducing the 5-10 methylene THF to 5 methyl THF, can also reduce biopterine Bq2 to B4 (29), [4]. Dihydrofolate reductase (21), besides reducing DHF to THF, can also reduce B2 into B4 [5, 6]. Folic acid is a vitamin which must be found in the diet. Biopterin (14, 28), on the contrary, is synthesised in our cells from GTP. Chemically the pteridine moiety is identical and those two molecules differ only by their lateral chain. In vitro, "THF" can replace B4 as cofactor of hydroxylases [75].

The cooperation [78] between the enzymes specific of each system cannot be taken as a mere coincidence. If such a built in salvage system exists, it shows how absolutely vital are the processes involved. Exactly as in rocket technology engineers double some very important parts, so that one could take over if the other fails, here nature seems to have realized the most fool-proof system possible.

All these safety measures explain maybe why mental retardation exists at all. If no supplementation existed, every break-down would produce complete neurological disaster, hence prevent any survival at all. On the contrary, thanks to the emergency measures, if one entry is blocked, some deleterious effect will result but sufficiently compensated so that only a diminution of the performances (and not a complete breakdown) would ensue.

Although the diseases enumerated in the legend of the figure 1 affect very different chapters of biochemistry their common effect is a severe trouble of monocarbon metabolism:

- By a diminished input of raw material, left column 1 to 7,

- or an imperfect transformation, 8 to 12,

- or an excessive consummation, 15 to 19,

- or a trouble in the complex pteridine regulating system, 14, 22, 28, 29,

- and its consequences even very remote, 37 to 42,

- or a shortage of reduced monocarbons, 23 to 27, and finally a lack of definite products 30 to 36.


The neurological approach

Indeed the fact that the vast majority of diseases producing mental retardation could be located on this diagram can hardly be accounted for by mere chance. But neuro anatomy tells us that the very structure of the brain explains this accumulation of catastrophes.

In order to build the eleven thousand millions of neurons of our brain, and to insulate with myeline the enormous wiring of the axones and dendrites (the length of which could extend from Paris to Mexico), an enormous production of monocarbons is necessary for the coded molecules (nucleic acids) as well as for various methylations. Moreover the production at the right place, right moment, and right amount of the security keys which open or shut the connexions, the synapses between neurons, necessitates also a full efficiency of the folic-biopterin systems.

If an experimental proof of these considerations is needed, it suffices to remember that antifolic drugs can produce incomplete closure of the nervous system [69] and folic acid during pregnancy can very effectively protect the children against these terrible diseases, spina bifida, anencephalia etc... [7, 8, 9]. The same is true with atresias of the digestive tract [10].

Remains a last term, not figured out in this scheme. Any logic-mimicking device invented by us requires the components (neurones), the insulation (the myeline), the efficiency of the gates (the yes or no of the synapses) but also an extremely refined wiring.

Inside the axones and the dendrites there exists a fantastically orderly network of very fine tubes, the neurotubules. They are considered by many authors as the real wiring of our brain. A network of a tremendous length. If put end to end all these neurotubules would go from here to the moon (and hopefully, back). Whether the constitution of the neurotubule network is directly affected by monocarbon metabolism is, for the moment, unknown. At least it is well established that this network is typically disturbed in Alzheimer [72] dementia and in a regressive Alzheimer-like syndrome so frequent in trisomic 21 patients. Also, the maturation of the neurotubule network is under control of thyroid metabolism [11], and deficiency of neurotubule network seems to be the cause of the mental retardation of untreated cretins [12] (athyreose or severe iodin deficiency). This role of neurotubules could possibly explain why neurones do not regenerate. The spindles of the mitotic apparatus and the neurotubules are made of the same building stones: the tubuline. Hence a cell has to choose definitely, either to keep assembling and disassembling tubuline for the mitotic machinery or to mount tubuline conservatively into an inner informative circuitry.


Clinical chemistry of trisomy 21


Uric Acid Excess and Purine Synthesis 17, 18, 19

The oldest known chemical trouble in trisomy 21 is possibly their slight excess of production and of excretion of uric acid [13, 14, 15].

This increased production of purine necessitates an over consumption of phospho-ribosyl and the first steps of its synthesis (shunt of hexose monophosphate) is accelerated in Down's syndrome [16].

The genes controlling the third (PRGS) the fourth (PRGFT) and the sixth (PRAIS) steps of the purine synthesis are on chromosome 21 [17,18] and explain this over-production of purines, finally excreted as urate.


The Lesch Nyhan Complication 15

Rarely very retarded trisomic 21 children, exhibit an irritable behaviour with autoaggressivity (beating their fingers, hurting their head) reminiscent of the automutilations in Lesch Nyhan syndrome [56].

In this terrible sex-linked disease, purines are spilling over because the salvage pathway (HGPRT) is deficient. The resulting over-consumption of monocarbons is probably the cause of the neurological damage.


The Homocystinuria Contertype 26, 27

If the enzyme cystathionine beta synthase does not function, homocysteine accumulates and is excreted as homocystine. The children affected by this disease are mentally retarded, have livedo reticularis (like trisomic 21) but are tall, slender, with long tapered fingers with some extra flexion creases. On the contrary trisomic 21 are of short stature have short fingers and frequently lack some flexion creases.

On this type-countertype effect, the hypothesis of a trouble of this reaction was put forward in 1975 [19] and demonstrated in 1984 [20]. In homocystinuria, excess of homocysteine blocks by competition the S-adenosyl methionine-methyltransferase. On the contrary in trisomy 21 the overburning of homocysteine impairs its remethylation in methionine, hence diminishing also the efficiency of the S-adenosylmethionine methyl transferases. Hence in both cases a deficiency of the various products of methylation.

It must be remarked that in trisomy 21 the furniture of 5-methyltetrahydrofolate is also impaired by the spilling over of monocarbons in the purine overproduction. Hence the deficit in cholinergic efficiency [21] and methylation of nicoti-namide [22], also noted in depressive illness [73].


Super oxide Dismutase Excess 3, 4

The metabolism of oxygen is impaired in trisomy 21. The enzyme superoxyde dismutase [23] is too active, so that too much of the 0-2 superoxide ion is destroyed and too much hydrogen peroxide H2O2 is produced. This toxic H2O2 is eliminated by glutathion peroxydase, the activity of which is increased in trisomy 21 [16].

Whether the diminution of the superoxide ion concentration could affect the tryptophane and 5 OH tryptophane indolamine oxydase or the synthesis of serotonine and 5HIAA via the biopterine system, or the synthesis of thyronine from tyrosine is presently unknown but may be worthy of investigation.


Hypothyroidy and Down's Syndrome 10, 20, 27

The association between the two diseases has been discussed for a long time and Bourneville was the first in 1904 to treat a recognized hypothyroidy in a trisomic 21 patient [24].

Since that time the discussion went over and Benda concluded from a series of autopsies that there was "exhaustion" of the thyroid among these patients [25].

Unfortunetly the recognition of the chromosomal basis of the disease slowed down the impetus of research in this field. Many reports quoted that hypothyroidy and to a less degree hyperthyroidy were too frequent in the disease [26, 27, 28] but Samuels et al. were the first to demonstrate in 1971 an excess of thyreostimuline hormone (TSH), soon confirmed by various authors [29].

The excellent work of Pueschel and Pezzullo 1985 [30] and the experimental data of Ozand et al. (1987) [31] confirmed formally the frequency of an elevated TSH and, seemingly a relative inefficiency ot T4 in Down's syndrome cells. Ozand even postulated some deficit of deiodination activity.

Independently we have systematically investigated thyroid function of our patients in Paris and found a highly significant elevation of TSH. On 52 patients the mean value is 4,59 ± 2,28, compared to 1,80 ± 0,87 in 51 controls (t = 8 for 101 d.f.).

Experimental work has shown an important effect of thyroxin upon the level of some enzyme activities. Thyroxine increases the 10-formyl THF synthase (9) and the 5-10 methylene THF reductase (20) thus increasing the input of oxydized monocarbons and the availability of reduced ones, 5-methyl THF, [32, 33, 34]. It also reduces the activity of 10-formyl dehydrogenase and diminishes the overflow (10), of CHO in CO2 [35] and diminishes the destruction of cystathionine and cysteine by cystathlonase (soluble cysteine desulphydrase) [36], (27).

All these effects favour the de novo synthesis of methionine hence favouring the methylation process. On the contrary, high doses of methionine diminish the 10 formyl THF synthase and directly excite the enzymatic efficiency of 10 formyi dehydrogenase [35]. These two regulations diminish the input of oxydized monocarbons.

Whether a fine tuning of TSH secretion and of T4 production by methionine and homocysteine levels exists is not known. But if it did, then the excess of cystathionine beta synthase should produce the elevated TSH and subnormal T4 of trisomic 21 patients. Also a possible regulatory role of reverse Ts, interfering with some enzymes of the folate pathway seems worthy of investigation.


Sensitivity to Methotrexate 22

As discovered by Peeters et col. in 1985, trisomic 21 children, when affected by leukemia, cannot suffer normal doses of methotrexate. This inhibition of dihydrofolate reductase causes toxicity at half of the dose tolerated by nontrisomic leukemic children [37]. We confirmed (Lejeune et al., 1986) on lymphocyte cultures that trisomic 21 are twice as sensitive as their normal sibs to mitotic index inhibition by methotrexate [38].

This sensitivity, now amply confirmed [39], must be compared to the production of encephalopathies in non-Down's Syndrome leukemics treated by methotrerate [74].

Using it as a tool it becomes feasible to test various additives to see if some substances can bring trisomic 21 cells back to a normal sensitivity. With Marie Peeters we are presently exploring the possible effects of various effectors and inhibitors. We already know [38] that methionine and homocysteine do not prevent the methotrexate toxicity.


The Heuristic Prospect

The present model of the pathogenesis of mental retardation in Down's syndrome is rather simple but encompasses most of what is known today. To summarize it briefly: There is a leakage of oxydized monocarbons due to the excess of purine production. Another leakage of reduced monocarbons corresponds to the increased turnover of cystathionine beta synthase [40] and is possibly related to difficulties of thyroid regulation. A slight deficit could also be related to the effect of superoxyde dismutase upon dioxygenases or various hydroxylases and reductases.

This would be in accordance with the serum level of dihydrobiopterin, higher in Down's syndrome [76] and lower in non-specific mental retardation in males [77].

Remains the association of Down's Syndrome and Alzheimer. The gene of familial Alzheimer's disease is possibly linked to chromosome 21 [41] and the gene controlling the precursor of amyloid substance is also in the 21 [42, 43). These facts could explain the relation between Alzheimer's disease and Down's Syndrome. Just as well the abnormal development of the neurotubules in the three related diseases: D.S., A.D. and hypothyroidy could point toward a common mechanism of these diseases; a propos, comparable lesions can be observed after head injuries [71, 72].

Medication trials with non toxic natural products interfering with monocarbon's metabolism can be conducted. Some are in progress and few indications emerge.

In case of severe regression pointing toward an Alzheimer-like evolution, substitutive medications enhancing tetrahydrofolate availability and methionine metabolism seem clinically promising. Also testing of the thyroid function is necessary and prudent supplementation with thyroid hormone in case of elevated TSH is clinically efficient.

No definite conclusion is at hand but the systematic investigation of the monocarbon metabolism remains a very urgent endeavour.



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