Fragile X Syndrome: A Possible Defect of Guanosine Pathway

Marie A. Peeters, Fabio Cattaneo, André Megarbane, Marie Odile Rethoré, Jérôme Lejeune

Brain Dysfunct 1992; 5:288-300


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Introduction

Fragile X syndrome is the second most common chromosomal cause of mental retardation and the most common cause of familial mental retardation. The mutation rate is high. The calculated incidence is 1/1,250 in males and 1/2,000 in females. Thus, accurate and early diagnosis is important for specific pharmacological and cognitive treatment.

Table 1. Patient characteristics
PatientSexAge yearsComplicationsTreatment mg/kg/dayDifferent treatment received mg/kg/dayClinical response
G.S.M6--FOL 0.9/INO 37/FOLIN 0.9+/0/+
G.L.M13--no drugs
H.C.M21--FOL 0.40
S.P.M12--FOL 0.7/FOLIN 0.90/+
B.V.F20Seizures-FOL 0.3+
P.J.M21--FOL 0.6/FOLIN 0.5/INO 32+/0/0
B.J.M17--FOL 0.6/FOLIN 0.9/INO 51+/0/-
B.R.M16--FOL 0.5/INO 51+/-
B.F.M9Psychotic-FOL 0.3/FOLIN 0.70
D.E.F7--no drugs
S.E.M19--no drugs
R.N.M10--FOL 0.9/FOLIN 10/+
B.C.M12--FOLIN 1.1+
R.V.M6Seizures-FOL 0.80
D.B.M16-FOL 0.3FOL 0.9+
T.A.M25Seizures-no drugs
B.G.M17-FOL 0.1/INO 27FOL 0.6/FOLIN 1/INO 27 + FOL+/+/+
M.B.M1--no drugs
A.S.M14Psychotic-FOL 0.8/FOL+INO 35+/0
P.O.F27Psychotic-FOL 0.9/INO 37+/0
H.M.F4--FOLIN 1.3-
P.G.M4Psychotic-FOLIN 0.86no data
D.B.M8--FOL 0.43+
G.F.M12--FOL 0.3-0.9/FOLIN 0.90/0
D.Y.M23Psychotic-FOL 0.6/INO 27+/0
D.A.M21--FOL 0.9/INO 290/0
M.M.M10--no drugs
J.O.M21--FOL 0.6/FOLIN 0.8/INO 150/0/0
Psychotic = self-aggressive or disruptive behaviour, stereotypes; FOL = folic acid; FOLIN = folinic acid; INO = inosine; + = beneficial, less agitated; o = no changes; - = regression, more agitated.

The syndrome is characterized by a marker X chromosome, identified by the presence of a fragile site on the distal long arm at Xq27. Mental retardation is transmitted in an X-linked dominant with incomplete penetrance pattern. Approximately 30% of the heterozygous female carriers are mentally retarded and about 20% of transmitting males show neither the fragile site nor clinical symptoms. Genotype and phenotype are therefore not absolutely correlated.

Sutherland observed that the expression of this fragile site in vitro depends on the composition of the culture medium and that deficiences of folic acid and thymidine promote expression of the fragile site in lymphocytes as does the addition of methotrexate [1]. This led to the hypothesis that folic acid may play a role in the pathogenesis of the mental retardation of this syndrome. Clinical trials using conventional doses of folic acid or high doses of folic or folinic acid do not seem to ameliorate the intellectual performances of these patients, but may have a positive effect on the behavioural manifestations [2-5]. A study of cells derived from fragile X patients and carriers seem to indicate that a primary folate defect is unlikely [6, 7].

Glover [8] and Tommerup et al. [9] independently showed that expression of the fragile site can be elicited by adding 5-fluoro-deoxy-uridine, an inhibitor of thymidylate synthetase, to culture medium containing folic acid but not thymidine. These observations suggest that perhaps some impairment in the de novo synthesis of thymidylate may play a role in fragile X syndrome. However, cellular thymidylate metabolism has been shown to be normal in cells with fragile X [10].

Much work has been done in vitro, modulating culture conditions needed to demonstrate fragile X expression [1,11-13]. In spite of all this work, the basic metabolic defect responsible for both fragile X expression and clinical manifestations remains unknown.

Recent work has focused on molecular research trying to locate the culpid genes found at the Xq27 locus. DNA research has been successful in unraveling the genetics of fragile X syndrome, but all remains to be done in understanding the pathophysiology of the disease. Both changes in methylation patterns and DNA fragment sizes have been shown in patients expressing the fragile X phenotype [14-19]. The condition is characterized by an inheritable DNA sequence that consists of an abnormal number of CGG repeats which are unstable during both meiosis and mitosis. Recently, a gene (FMR-1) has been identified which contains the unstable DNA region and which is not expressed in most fragile-X-affected males [20, 21].

Gene localization and sequencing does not necessarily mean understanding of global cellular metabolic dysfunction and the patho-physiological consequences thereof, thus leading to comprehension of how the gene abnormality produces disease. One might therefore be peering down the wrong end of a telescope.

We have, in recent years, been investigating the hypothesis that a defect of purine metabolism, be it primary or secondary, may play a major role in mental retardation syndromes [22-24].

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Material and Methods

Twenty-eight peripheral blood samples from patients with documented fragile X syndrome (24 males and 4 retarded females) were cultured for 72 h in TC199 medium (Seromed) supplemented with 25% human AB serum, phytohaemagglutin C (IBF France), penicillin and streptomycin. Culture technique, harvesting and microscopic examination were carried out according to the previously described method [22]. Patient characteristics are shown in table 1.

The following products were added on initiation of culture (all products were Sigma products unless otherwise specified); the entire panel could not always be performed for each patient: (1) inosine 125 mg/l; (2) alanosine 0.0625 mg/l, kindly provided for by the Laboratory of Biochemical Pharmacology (National Institute of Health, Bethesda. MD, USA); (3) aza-serine 0.0156 mg/l; (4) adenosine 16 mg/1; (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 10-6 M; (10) theophylline 750 mg/l.

The lymphocyte cultures were harvested with the standard techniques used for chromosomal analysis. Slides were stained with Giemsa and coded. A minimum of 3,000 cells were read by two different observers to calculate the mitotic index, expressed as the ratio of mitosis over the total number of nuclei (22].

Table 2. Percentage increment or decrease of mitotic index in patients with fragile X syndrome, controls and patients with mental retardation of other etiologies
GuanosineTheophyllineAzaserine
Total Xqfra patientsN192126
M18.4-28-3
SD14.62019
Total patient sampleN215108237
M2.8-130
SD24.328.422
t2.742.3NS
P0.01<p<0.0050.02-
Total control sampleN803281
M5.61.73
SD24.427.521
t2.173.8NS
P0.05 <p< 0.025<0.001-
Trisomy 21N341338
M-21.74
SD30.635.930
t2.683NS
P0.010.005-
Mental retardationN582861
M-2.6-20-2
SD20.427.520
t4.11.2NS
P< 0.001--
Results were analyzed by comparing the values observed in fragile X patients with those obtained in dif-ferent patient populations. N = number of patients; M = mean mitotic index (increase or decrease); SD = standard deviation; t = Student's t test.

Twenty-six patients were on no medication at the time of the lymphocyte culture.

Controls used for the analysis were as follows: 135 normal adults, 79 patients with trisomy 21,10 patients with cri-du-chat syndrome and 68 patients with mental retardation (with or without a chromosomal anomaly other than the ones mentioned above).

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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 were expressed as the percentage increment or decrease in mitotic index. Statistical comparisons between groups were based on Student's t test (two-tailed test).

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Results

As is shown in tables 2-4 (only data relevant to the present study are shown), there were three products which, when added to the lymphocyte culture, altered the mitotic index in a significant and specific manner.

Table 3. Percentage increment or decrease of mitotic index in males with fragile X syndrome, male controls and patients with mental retardation of other etiologies
GuanosineTheophyllineAzaserine
Fragile X malesN161824
M17.6-27-3
SD15.720.919
Total male sampleN9058117
M-1.4-20-1
SD21.82520
t3.3NSNS
P0.001--
Normal male controlsN291228
M-1.5-5.82
SD192719
t3.352.5NS
P0.005 <p< 0.0010.025-
Trisomy 21 malesN17519
M-0.51.49
SD25.533.316
t2.362.22.2
P0.0250.05 <p< 0.0250.05 <p< 0.025
Retarded malesN341936
M-2.8-26-3
SD2319.122
t3.15NSNS
P0.005 <p< 0.001--
Results were analyzed by comparing the values observed in fragile X male patients with those obtained in different male patient populations. Symbols are as in table 2.

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Guanosine

The addition of guanosine increased the mitotic index significantly in patients with fragile X syndrome. When compared to the total patient sample, patients with mental retardation of various etiologies and patients with Down's syndrome, the difference was highly significant (0.01 < p < 0.005); when compared to the total control sample the difference was not as marked but remained significant (0.05 < p < 0.025). On analysis, this seemed to be due to differences between male and female response to guanosine (female controls increasing their mitotic index significantly more than male control patients: p = 0.025). We therefore broke down the analysis to examine response only in male patients. As is shown in table 3, there was a highly significant difference in the response to guanosine when fragile X males were compared to the total male sample (p = 0.001), normal male controls (0.005 < p < 0.001) or retarded males of other etiologies (0.005 < p < 0.001). The difference between fragile X males and males with Down's syndrome was significant (p = 0.025).

Table 4. Percentage increment or decrease of mitotic index in patients with fragile X syndrome and in patients with Down's syndrome
GuanosineTheophyllineAzaserine
Total Xqfra patientsN192126
M18.4-28-3
SD14.62019
Trisomy 21, psychotN828
M13.4-12
SD31.3-20
tNS-2
P--0.05
Trisomy 21, non complN12614
M-14-7.23
SD21.126.122
t4.942NS
P<< 0.0010.05-
Fragile X malesN161824
M17.6-27-3
SD15.720.919
Trisomy 21, psychot, maleN7-7
M20.1-9
SD27.5-19
t0.3-1.5
Trisomy 21, non compl, maleN747
M-19-1515
SD7.39.312
t5.621.12.3
P<< 0.001-0.025
Results are analyzed by comparing the values of the mitotic index in fragile X patients and those obtained in Down's syndrome patients. Symbols are as in table 2. Psychot = psychotic complications; no compl = uncomplicated Down's syndrome.

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Theophylline

We observed a significant decrease in the mitotic index in patients with fragile X syndrome when compared to the response observed in the total control sample (p < 0.001) and in the trisomy 21 sample (p = 0.005).

However, there was no difference between the response observed in fragile X syndrome patients and patients with mental retardation. When the response in the male population was analyzed, a difference was observed between normal male controls and fragile X males (p = 0.025); none of the other categories studied showed a difference in response. Moreover, the sex difference which had been noted between normal males and females in their response to guanosine was not observed with theophylline.

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Azaserine

Azaserine is a glutamine antagonist inhibiting numerous steps of nucleic acid synthesis, the most sensitive reaction being the inhibition of phosphoribosylformylglycineamidine synthetase. A slight increase in the mitotic index was observed in male patients with Down's syndrome, the difference in response was significant when compared with that observed in fragile X males (0.05 < p < 0.025). Further analysis was difficult given the small sample size. This difference was observed only with Down's syndrome patients, not with normal controls.

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Changes in Mitotic Index

To test whether the changes observed were specific for fragile X syndrome, we looked for patients manifesting an increment in mitotic index of more than 10% (over their baseline) in the presence of guanosine: (1) 13/19 patients with fragile X showed an increment of over 10% in the presence of guanosine (all fragile X patients increased their mitotic index); (2) 29/80 normal controls (?2 = 6.5; 0.02 < p < 0.01); (3) 7/29 normal male controls (?2 = 9.3; 0.01 < p < 0.001); (4) 12/34 patients with trisomy 21 (?2 = 5.4; p = 0.02), 7 females and 5 males (all 5 males had psychotic complications); (5) 17/58 patients with mental retardation (?2 = 9.2; 0.01 < p < 0.001), 7 females and 10 males. It is of note that 9 of the 10 male patients had associated psychiatric complications. Seven males showing an increase in mitotic index in the presence of guanosine had originally been referred for Xqfra investigation but had been found to be Xqfra negative on cytogenetic investigation.

In our whole sample, 72 persons were tested for both theophylline and guanosine reactions. Of these 72, 18 showed the association: increment of over 10% in the presence of guanosine, decrease of greater than - 15% in the presence of theophylline, 7 were fragile X patients, 2 were parents of fragile X patients, 2 were patients with X-linked mental retardation syndrome (at least 2 affected male sibs). Therefore, 11/18 (73%) of the positive sample were related to fragile X syndrome. The remaining 7 patients (9%) were: 4 normal female controls, 2 patients with mental retardation and 1 patient with Down's syndrome.

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Discussion

Attempts to explain our findings and to correlate them with the mechanisms responsible for the various neurologic manifestations are hampered by the inexistence of data on the neurochemical and purinergic derangements in fragile X syndrome. We will therefore review what is known about the biological effects of both guanosine and theophylline and advance hypotheses as to how our observations could tie in with both known effects and clinical findings in fragile X. We will then review drug trials which have been tried in fragile X syndrome and how the results correlate with our observations.

We found only one study examining the effect of guanosine on the expression of the folate-sensitive fragile site [11]. Although inhibition of fragile site expression was noted, the culture medium (thymidine deficient), as well as the guanosine dose used (30 times greater) were different from ours, rendering any useful comparison impossible.

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Guanosine and Thyroid Studies

Thyroid function tests in fragile X syndrome males and in X-linked mental retardation males have shown normal basal serum levels of thyroid hormones and thyroid-stimulating hormones (TSH) but a blunted TSH response to thyrotropin-releasing hormone (TRH) [25, 26]. Since TRH receptor binding may be allosterically regulated by guanine nucleotides [27], one might hypothesize that, if fragile X males have some defect along the guanine nucleotide pathway, this could alter TRH receptor binding and thus TSH response. These observations are in keeping with the beneficial effect of guanosine reported here.

We recently published our observation suggesting a relationship between TSH and reverse T3 in Down's syndrome [28]. Although the metabolic action of reverse T3 is not known, we have suggested that reverse T3 may be a modulator of inosine monophosphate dehydrogenase [29], the precursor of guanosine monophosphate (GMP). Reverse T3 levels have never been studied in fragile X syndrome, but given our observations, it would be well worth an investigation.

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Theophylline, Purine Metabolism and Autism

There are numerous studies suggesting that fragile X is related to autism (an association which could be as high as 43%), but this is not universally admitted [see 30 for review]. Although the biochemical defect responsible for autistic behaviour is not known, it is worth remembering that two interesting biochemical abnormalities have been reported in relation to autism [31, 32]. Inborn deficiency of adenylosuccinate lyase gives rise to a clinical picture rather similar to autism [31]. Furthermore, autistic children have been found to have elevated serum cyclic adenosine monophosphate (cAMP) levels [see 32 for review]. These findings point to some defect or impairment along the adenosine metabolic pathway.

Theophylline and other methylxanthines can produce clinical changes which are not unlike those observed in patients with fragile X syndrome: anxiety, restlessness, hyperactivity and behaviour abnormalities [33].

Theophylline is a non-selective antagonist of adenosine receptors (both Al and A2). It is also a known phosphodiesterase inhibitor (which increases cAMP intracellular levels). The decrease in mitotic index observed in fragile X males, which is significantly different from the response observed in normal controls could therefore indicate some degree of impairment in DNA synthesis which could be due either to adenosine deficiency or imbalance in adenosine-guanosine nucleotide (and/or cAMP/cGMP) ratio.

It has been shown that substances which elevate cAMP levels (as does theophylline by virtue of inhibition of phosphodiesterase) can simultaneously cause a decrease in cGMP levels [34]. If fragile X patients already have some impairment along the guanine nucleotide metabolic pathway, any further impediment would be extremely detrimental and could explain the significant decrease in mitotic index which we observed in these patients, when theophylline was added to their lymphocyte cultures.

The possible association between autism and fragile X syndrome, the observation that autistic patients have increased cAMP levels, the fact that theophylline increases cAMP levels and our observation that theophylline decreases the mitotic index in fragile X constitute an interesting and coherent observation.

If one tries to tie together both the apparently beneficial effect of low-dose guanosine with the theophylline toxicity, one might hypothesize that the metabolic defect in fragile X patients lies somewhere in the complex interaction between guanosine and adenosine, either by virtue of nucleotide pool imbalances or at the receptor level. It is of great interest that theophylline treatment has been found to have little action on the A1 receptor number but may alter the coupling between Al receptors and regulatory guanosine-5'-triphosphate (GTP)-binding proteins [35]. In rat brain there are regional differences in the effects of GTP on adenosine A1 receptor binding [36]. Both these observations have implications for future research.

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Guanosine: The Brain and Mental Retardation

In recent years, the association between purine metabolic disorders and neurological dysfunction has been reported in a variety of genetic disorders [24, 37], opening new and exciting research perspectives.

The effect of guanine nucleotides on central nervous system neurotransmitters is exceedingly important. It is not known whether guanosine is a neurotransmitter, as is adenosine, but guanine nucleotides are known to negatively modulate agonist binding to adrenergic, dopamine, muscarinic cholinergic and opiate receptors [38, 39]. Phencyclidine receptors are also regulated by guanine nucleotides [40]. Furthermore, guanine nucleotides play a major role in pterin metabolism (GTP is the precursor of tetrahydrobiopterin), as well as in microtubule dynamics [41].

Both cAMP and cGMP play a unique role in neurotransmitter regulation, and their ratio seems to be of clinical importance. Indeed, it has been suggested by Goldberg et al. [42] that these two cyclic nucleotides function together via dual opposing effects regulating the cholinergic-adrenergic balance (the Yin-Yang hypothesis). An imbalance between cAMP and cGMP has been reported, for example, in Lesch-Nyhan syndrome [34].

After birth, purine metabolism in the central nervous system is characterized by reduced de novo purine synthesis, increased hypoxanthine-phosphoribosyltransferase activity and absent xanthine oxidase activity [43]. Therefore, the brain is largely dependent on salvage pathways to sustain GTP levels. The switch in purine metabolism from essentially de novo during brain development to via the salvage pathway after the main burst of neuroblast and neuroglial proliferation might explain why patients with fragile X have essentially normal cerebral morphology, the main neuro-anatomical change documented in these patients (as well as in patients with autism) being hypoplasia of the posterior cerebellar vermis [44, 45]. This situation is therefore quite different from cri-du-chat syndrome, in which we suggested a defect in de novo purine synthesis and which is associated with significant morphological changes of the brain [24, 46].

Any metabolic defect along the purine salvage pathway would have neurological and behavioural consequences manifest after this period of neuroblast and neuroglial proliferation. The perinatal period during which gene switching and receptor imprinting occurs is sensitive to late 'metabolic teratogenesis' [47]. A genetic, metabolic defect involving a gene or group of genes normally activated during this period will have important repercussions on membrane receptor imprinting and postnatal brain development. It is of interest that the analysis of the neocortex in fragile X adults showed abnormal dendritic spine morphology suggesting an anomaly of postnatal dendritic maturation [48, 49].

Our findings suggest that fragile X patients have some metabolic anomaly along the guanine pathway, an observation which has important implications. Given the complexity of guanosine metabolism, of the interactions between the different nucleotide pools as well as ofpurinergic neuromodulation, further research is required for comprehension of the pathophysiology of fragile X syndrome.

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Medication Used in Fragile X Syndrome and Possible Guanosine Deficiency

Folate has been found to inhibit GTPase activity and stimulate the release of guanosine-5'-diphosphate from brain membranes (greatest in cerebellar and hippocampal membranes) [50]. These findings are interesting, since folate therapy has been found, in a few studies, to be beneficial in fragile X patients. The fact that cerebellar and hippocampal anatomical anomalies have been reported in patients with autism and that cerebellar anomalies are known in fragile X patients is also noteworthy [44, 45, 51]. The link between guanine nucleotide metabolism, cerebellar function, fragile X and autism seems worth to be further investigated.

Stimulant medication (methylphenidate and dextroamphetamine) which inhibit the synaptic re-uptake of dopamine and noradrenaline has been reported to be beneficial for some patients with fragile X syndrome [52]. Imbalance between the cholinergic and dopaminergic systems has been implicated in the etiology of several neurological and psychiatric disorders, and both these systems are modulated by cGMP [38].

Several drugs have been found to modulate cGMP levels and/or alter cAMP/cGMP interaction: haloperidol, lithium and carbamazepine [53 - 56]. It is of note that these drugs have been reported anecdotally to be beneficial in some cases of fragile X syndrome [57].

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Conclusion

Given our findings, which suggest that fragile X patients have a metabolic defect along the guanine nucleotide pathway, further studies on the biochemical defects in these patients seem promising. Since this pathway plays a major role in both neurological and psychiatric disorders, studies allowing the pinpointing of the exact metabolic defect as well as investigations on membrane receptors, cyclic nucleotides, possible neurotransmitter deficiencies or imbalances should be performed not only in patients with fragile X syndrome but also in patients with X-linked mental retardation. Better understanding of the biochemical defects is mandatory for comprehension of the pathogenesis and is a prerequisite for designing possible therapies for these patients.


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