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Cyclic AMP metabolism in fragile X syndrome.

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Cyclic AMP Metabolism
in Fragde X Syndrome
Elizabeth Berry-Kravis, MD, PhD, and Peter R. Huttenlocher, M D
Cyclic AMP (CAMP)metabolism was studied in platelets from a series of 14 patients with fragile X syndrome (fra X)
and 2 1 control individuals. 1-Isobutyl-3-methylxanthine
was used to inhibit phosphodiesterase and thus measure CAMP
production, prostaglandin E, was used to assess receptor-mediated cAMP accumulation, and forskolin was used to
directly stimulate the catalytic subunit. In patients with fra X, basal production was 63% of that of control subjects
( p = 0.019). Prostaglandin El- and. forskalin-stimulated production were 61% ( p = 0.039) and 56% ( p = 0.012) of
that of control subjects, respectively. cAMP production in 8 patients with fra X overlapped the control range, whereas
measures of production in 6 patients formed a cluster with values lower than any of the 2 1 control subjects assayed,
suggesting possible biochemical heterogeneity within patients with fra X. Results obtained from the group of patients
with fra X suggest possible abnormal function or regulation of the catalytic subunit of adenylate cyclase in at least a
subgroup of patients with fra X. Variability of biochemical findings in patients with fra X may reflect the known
high variability of the clinical syndrome.
Berry-Kravis E, Huttenlocher PR. Cyclic AMP met;tbolism
in fragile X syndrome. Ann Neurol 1992;31.22-26
Learning and short-term retention ‘of information are
thought to depend on activation of intracellular second
messenger systems that, in turn, activate protein kinases responsible for phosphorylation of substrate proteins capa.ble of mediating modification of synaptic
properties. One such second messenger system is the
cyclic AMP (CAMP) cascade. Receptors bind neurotransmitters or other neuroactive ligands to activate the
cascade, causing interaction of receptor proteins with
regulatory subunit trimers (G, or Gi for stimulatory or
inhibitory, respectively) of adenylate cyclase. Regulatory subunits then interact with the ciatalytic subunit of
adenylate cyclase to increase o r decrease production of
CAMP. lntracellular cAMP activates (:AMP-dependent
protein kinases resulting in phosphorylation and activation of (or deactivation of) their protein or enzyme
substrates, which regulate synaptic fu.nctions necessary
for short-term neuronal memory. Phiosphodiesterases
inactivate cAMP by hydrolysis, and phosphatases dephosphorylate protein substrates (for reviews, see 11,
Biochemiical lesions of this cascade would logically
produce learning or memory deficits if the intact cascade is truly required for these processes. In fact, lesions of the cascade in several lower organisms and
even in humans do appear to produce “learning” problems. In ApIysZu. blockade of cAMP production or in-
From the Departments of Pediatrics and Neurology University of
Chicago, Chicago, IL.
Received Feb 18, 1991, and in revised form Jim 4. Accepted for
publicarion Jun 5, 199I .
jection of inhibitors of CAMP-dependent protein
kinases prevents short-term sensitization of the gill
withdrawal reflex 131. Learning-deficient Drosophila
mutants have biochemical lesions at multiple levels of
the cAMP cascade 141, including the catalytic subunit
(ra&zga mutant) and the G, regulatory subunit (t,umip)
of adenylate cyclase and phosphodiesterase (ahme).
Humans with type la pseudohypoparathyroidism
(PHP) have been shown to have diminished activity
of the G, regulatory subunit of adenylate cyclase
{ 5 ] and various mutations within the G, gene [GI, Although patients with other forms of PHP have similar
problems with calcium metabolism, only those with
type Ia have learning deficits and mental retardation
171, presumably because of impaired cAMP cascade
function in nervous tissue.
The fragile X syndrome is a common mental retardation syndrome, associated with a folate-sensitive fragile
site at Xq27.3. The phenotype is quite variable and
patients demonstrate intelligence levels that rmge
from normal to severely impaired { 8 ] . Other associated
features are also variably present and include autisticlike behaviors, facial dysmorphism, large ears, large testicular size, hyperextensible joints, and dermatoglyphic
abnormahties. Patients with fragile X syndrome have
particular problems with short-term memory and sequencing of new information [9}, which might be the
Address correspondence to D r Berry-Kravis, Section of Pediatric
Neurology, University of Chicago, 5841 South Maryiand Avc, Box
228, Chicago, 1L 60637.
Copyrighi: 0 1992 by the American Neurological Association
sort of functions most affected by a cAMP cascade
lesion. Further, although the gene that apparently mediates mental retardation in fragile X syndrome has
recently been identified {lo, 111, neither the function
of the gene product nor the mechanism through which
its absence produces cognitive dysfunction is currently
Materials and Methods
Prostaglandin El (PGE,), forskolin, 1-isobutyl-+methylxanthine (IBMX), and cAMP antisera were obtained from
Sigma Chemical (St Louis, MO) [1251}cAMPderivative for
radioimmunoassay (RIA) was obtained from ICN Radiochemicals (Irvine, CA).
Patients with fragile X syndrome were patients recruited
from the Pediatric Neurology Clinic at the University of Chicago (Chicago, IL). All patients (age range, 5-50 yr) had
documented fragile X syndrome both clinically and by cytogenetic testing, demonstrating the fragile site at Xq27. There
was considerable clinical variability. Intelligence ranged from
borderline to severe mental retardation, and expression of
facial dysmorphism was also highly variable. Most patients
were not taking medication, however, 2 patients were treated
with methylphenidate hydrochloride, 1 with valproic acid,
and 1 with ethosuximide. No patients were receiving folate
treatment at the time of the study. The patient on ethoswimide was assayed both on and off the medication with no
significant difference in assay results. Control subjects were
normally intelligent individuals (age range, 15-60 yr) who
were free of illness and taking no medication. Some mentally
retarded (non-fragile X) patients in the same age ranges
were studied as mentally retarded control subjects. Blood
was obtained from patients and control subjects at the same
time and drawn into syringes containing 1/10 volume 3.8%
citrate anticoagulant.
Platelet Isolation
cAMP metabolism was measured using a platelet assay system because adenylate cyclase activity is highly expressed in
platelets that, like brain, use several second messenger systems to modulate responsiveness [ 12). Additionally, platelets
are conveniently isolated from blood samples and contain
receptors that stimulate (PGE,) and inhibit (a-adrenergic)
adenylate cyclase. Platelets were isolated from blood samples
by modifications of previously described methods [13, 14).
Blood samples were subjected to centrifugation at 400 g and
the platelet-rich supernatant removed. Packed erythrocytes
were then washed with a glucose-containing phosphatebuffered balanced salt solution twice followed by centrifugation at 400 g. The combined platelet-rich supernatant fractions were centrifuged at 5,000 g and the platelet pellet was
washed twice. Platelet pellets were then resuspended in
glucose-containing phosphate-buffered balanced salt solution
at p H 7.5 at a concentration of 0.25 to 0.50 mg/ml.
Metabolic Studies in Intact Platelets
Platelet suspensions were added to triplicate polypropylene
tubes in 2 0 0 - 4 volumes. Appropriate drugs were added to
each tube in 10-pl volumes to give a final reaction volume
of 220 p1. IBMX was used to inhibit phosphodiesterase.
Drugs were prepared freshly for each experiment (PGE,)
or diluted from concentrated stock solutions kept frozen or
refrigerated (forskolin, IBMX). Reactions were incubated at
35°C for 30 minutes and then stopped by addition of 0.5 ml
of 8% trichloroacetic acid (TCA). Sedimented protein was
separated by centrifugation at 10,000 g for 10 minutes. The
CAMP-containing supernatant was extracted with 2 ml of
water-saturated ether four times to remove TCA. Extracted
samples were dried under air and stored at 0 to 4°C. Protein
was quantitated by the method of Lowry and colleagues [ 151.
cAMP was quantitated by RIA [16].
Five patients with P H P and mental deficiency were studied
to document that the assay system could identify known defects in cAMP metabolism. Basal cAMP production was not
statistically different (85 2 11%) from control values (mean
SEM) but PGE,-stimulated cAMP production was only 22
t 5% of same-day control values, consistent with the known
G, deficiency [5] and the resultant defect in receptormediated stimulation of cAMP production in these patients.
The platelet preparations used had intact regulation of
cAMP levels. Exposure to PGE, resulted in a dosedependent receptor-mediated 20- to 40-fold elevation
of cAMP production. Forskolin, acting through a
G,-mediated mechanism as well as through direct action on the catalytic subunit in intact platelets [17],
gave a dose-dependent 40- to 60-fold increase in production. A small (approximately three-fold) increase in
basal cAMP level was seen when IBMX was added to
inhibit phosphodiesterase. This increase (CAMP level
in IBMX minus basal cAMP level without drugs) is a
measure of basal cAMP production.
cAMP production in platelets from a series of 21
control subjects and 14 patients with fragile X syndrome is shown in the Table. Basal cAMP levels were
not significantly different between control subjects and
patients with fragile X syndrome. When IBMX was
used to inhibit phosphodiesterase, basal production
(level in IBMX minus basal level) was significantly
lower in fragile X samples. cAMP production in PGE,
and in forskolin was also significantly lower in the
group of patients with fragile X syndrome. The means
for all measures of cAMP production in the fragile X
group were about 60% of control means. Similar results were obtained when data were analyzed including
only the 9 patients with fragile X syndrome who were
not on medication (see Table). Although measures of
cAMP production were slightly lower in the unmedicated group, differences are not statistically significant
and probably do not represent a true effect of the medications on the assay. There was no effect of sex on
cAMP production (in forskolin, cAMP production in
control males [n = 101 was 11,540 5 2,270 and in
females [n = 111, 11,080 2 1,760; mean 5 SEM).
There was also no significant correlation between age
Berry-Kravis and Huttenlocher: Cyclic AMP in Fragile X
Cyciil- AMP Productma in Patients with Fragile X Syndrome and Control Sihjecti
cAMP (prnolirng of protein)
Control S u b j e c t s
(n = 21)
Basal level
IBMX alone
Basal production
PGE, (10 KM) + IBMX
FSK (50 pM) + IBMX
75 ?
145 ?
6,850 i11,300 t-
* 30
Fra X
( n = 14)
Fra X"
(n = 10)
6'5 t 11
146 ? 18
81 i 14
4,300 _t 800
6,900 i: 1,400
58 i 12
126 & 23
7 1 i 17
3,600 ? 1,000
6,000 ? 1,800
Fra Xlc
Fra X " l c
('% )
0.49 5
0.0 1 I
Cyclic AMP (CAMP)wa!i measured as described in the text and is given in picomoles per milligram of protein. T h e concentration ( i f prosraglaniliri
E, (PGE,) was 10 pM and forskolin (FSK) was 50 pM. Valui:s represent the mean 2 SEM. Basal production is ciehneci as the cAMP level in
1-isobutyl-3-methylantiline (IBMX) minus the basal level of CAMP. Values of p are calculated with respect to the control group and werc.
determined with Student's t test. Fra X refers to the group of all patients with fragile X syndrome and Fra Xa refers t o the group of patienth
with fragile X syndrome who were not treated with medication
c = control.
and any measure of cAMP production in either the
control or the fragile X group (for basal production: Y
= 0.032, p = 0.89 {control subjects] and Y = 0.087,
p = 0.77 [patients with fragile X syndrome); for
forskolin-stimulated production: r = 0.047, p = 0.841
[control subjects) and Y = 0.247, p = 0.395 [patients
with fragile X syndrome)). Six patients with nonspecific
mental retardation (not fragile X) have additionally
been studied and demonstrated cAMP metabolism indistinguishable from same-day control subjects.
The distribution of values for PGE,- and forskolinstimulated cAMP production in the series of patients
and control subjects shown in the Table is plotted in
Figure 1. As can be seen from the plot, approximately
50% of the patients with fragile X syndrome demonstrate cAMP production falling within the control
range. A cluster of patients clearly have lower cAMP
production in the presence of either agent than a n y
control subjects assayed. The same patients in whom
cAMP production was below the control range in
PGE, also had low values with forskolin. Six of the
determinations on individual patients with fragile X
syndrome have been repeated two to three times on
different blood sarnples with similar results, that is,
patients falling in the control range do so in the repeat
assay and patients in the low cluster persistently assay
lower than the control range. Additionally, several of
the control individuals have been repeated numerous
times with high reproducibility of values over multiple
different assays.
Typical dose-response curves for cAMP production
in PGE, and forskolin are shown for a patuent with
abnormally low CAMPproduction and a same.-day control subject in Figure 2. This patient has demonstrated
reproducibly low cAMP production with respect to the
control group and with respect to control subjects
done o n the same day The shapes of the doseresponse curves are similar in patients with abnormal
metabolism and control subjects, suggesting diminished maximal cAMP production rather than clecreased
Annals of Neurology
Vol 31 No 1 January 1992
(pmi 15000
fra X
fr., Y
Fig 1 . Range of CAMPproduction in a series of patients with
fragile X .syndrome and control indifiduals assayed in the same
experiments. CAMPproduction is shown as mea.izrred in I B M X
with 10 pM PGE, (A) or in IBMX with 50 pM forskolin
(Bi. Data are plotted a1 picomoles of cAMP produced per milligram of protein. All determinations were done in triplicate.
When individuals were assayed twice, data pointi represent
averageJ of the two determinationj
potency of reagents. These dose-response curves arc
typical examples of curves obtained from platelets of
all patients with below-normal cAMP production.
In this preliminary study, we provide evidence that
some patients with the fragile X syndrome may have
9 ~ a
Fig 2. Dose responses of cAMP production t o PGE, and forskolin (FSK). CAMPproduction in platelets of a representative
patient with fragile X syndrome (A)and the same-day control
are plotted in the presence of IBMX and inindividual (0)
creasing doses of PGE, (A)and FSK (B). Data are plotted as
picomoles of CAMPproduced per milligram of protein. All determinations were done in triplicate.
a defect in CAMPproduction in a platelet assay system.
Our data also suggest that such biochemical heterogeneity may exist. Alternatively, the population with fragile X syndrome may represent a normal distribution
that overlaps the distribution in control subjects. A
larger study group will be required to resolve this issue.
Comparison of biochemical and IQ data in this series
of 14 patients does not show a statistically significant
correlation. If two very atypical, profoundly retarded
(IQ(20) individuals (one with intractable seizures and
a Lennox-Gastault pattern on electroencephalogram)
are eliminated from the analysis, however, a statistically
significant correlation is seen ( r = 0.58; p = 0.05).
Larger numbers of patients will be needed to definitively establish whether a correlation exists. Additionally, correlation of scores on neuropsychological tests
specifically involving memory and sequencing, behavioral manifestations, and other aspects of clinical phenotype with results of biochemical testing are under
way. If such correlations are found, some assessment
of the role of the cAMP cascade in the complex process of human learning and memory may be possible.
A number of factors may, however, complicate analyses of clinical and biochemical correlations and contribute to apparent biochemical heterogeneity within
fragile X syndrome. These include potential differences in adenylate cyclase expression between tissues and individuals. Because basal production and
forskolin-stimulated cAMP production are affected as
much as receptor-mediated stimulation of cAMP production, the fragile X data are most suggestive of a
partial defect in function of a catalytic subunit of adenylate cyclase. This abnormality might be related to a
mutation in a structural or regulatory gene for a catalytic subunit itself or to an abnormality of a protein or
membrane component that influences cyclase activity.
Four catalytic subunits are now known in mammalian systems. These cyclases appear to be expressed
differentially in tissues { 181. Although the abnormal
catalytic subunit in the Dvosophda learning mutant rutabaga is X-linked [4],no mammalian catalytic units have
yet been mapped to a chromosome. It is possible that
if only one of several cyclases is partially affected in
patients with fragile X syndrome (as in rutabaga where
only one of two cyclases is partially affected and
calmodulin-responsiveness appears to play a key role
in learning [19}), only part of the activity in any given
cell would be affected, leading to less evident differences between control subjects and patients and possible overlap between control and patient data ranges.
Differential tissue expression in different individuals
might lead to some of the variability in the control
group in addition to the appearance of subgroups in
the patient data set even if all patients had identical
mutations. A cyclase less expressed in platelets might
be a major cyclase in brain, giving a biochemical defect
small enough to be insignificant for function in platelets, but large enough in brain to produce learning difficulties. Alternatively, such a cyclase might be developmentally regulated in brain and mediate processes
important for neuronal development or organization.
Another factor that may complicate clinicobiochemical correlations is potential variation in expression of
the genetic abnormality between fragile X tissues and
individuals. A two-step mutation process appears to
explain unusual fragile X inheritance patterns such
as transmission of the syndrome through unaffected
males C201. This process involves an initial mutation
that predisposes an area of the chromosome at Xq27
to a subsequent regional defect in reactivation in a cell
that will then pass through oogenesis in the female (the
second step in the mutation process). The cells that
contain the reactivation error then carry an “imprinted”
fragile X chromosome, which is responsible for expression of the fragile site and the clinical syndrome. Several groups have recently shown hypermethylation of
D N A fragments from near the fragile site in mentally
retarded males with fragile X syndrome [lo, 111, consistent with the concept of regional inactivation. It has
been shown that many patients with fragile X syndrome are actually mosaic for the primary mutation,
the imprint, and FMR-1 MRNA expression C21f.
Thus, varying portions of cells in any tissue would be
expected to express the biochemical defect resulting in
a high degree of interindividual and intertissue variability. This might explain some of the variability seen in
our assay as well as the large variability in fragile site
expression between tissues {22). Also, cognitive function would not necessarily correlate with fragile site
percentages or metabolic studies in tissues other than
brain. Indeed, no correlation is seen between fragile
site percentage in lymphocytes and IQ { S ] .
It will be possible to test whether biochemical heter-
Berry-Kravis and Huttenlocher: Cyclic AMP in Fragile X
ogeneity in cAMP production in fragile X tissues is, in
fact, related to fragile site expression by using easily
accessible tissues that can be growri in culture, such as
lymphoblastoid cells and fibroblasts. These cultures
will also allow for study of cAMP merabolism abnormalities in multiple tissues free of variables such as
medication and diet, and will provide a tissue source
for enzyme (adenylate cyclase) assays, protein purification, and genetic analysis. If abnormal cAMP metabolism can be confirmed in cell cultures, such studies will
be necessary to establish specific enzvme defects or
interactions responsible for diminished cAMP production in fragile X samples, and their relationship to the
mutations in fragile X syndrome.
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