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,
2)). 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 .
22
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 known.
Materials and Methods Materials 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 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.
*
Results 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
23
Cyciil- AMP Productma in Patients with Fragile X Syndrome and Control Sihjecti
cAMP (prnolirng of protein) Reagents
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 ? 220 145 ? 6,850 i11,300 t-
16
* 30
17 660 1,400
Fra X ( n = 14)
Fra X" (n = 10)
(57)
P
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
87 66 63 61 56
0.651 0.0: 0.019 0.039 0.012
__
-
Fra Xlc
Fra X " l c ('% )
P
77 57
0.49 5 0.053
40
0.0 1 I 0.000 0.032
53 53
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 24
Annals of Neurology
Vol 31 No 1 January 1992
A 14000
R
A
3ouoo
A
12000
25000
loo00
zooom
f 8000
CAMP @mi ms) 6000
A
A A
A
A
A
A CAMP (pmi 15000 mg)
i
f
t
4 A A
A
f
4 10000
A 4
f
4000
'
A
f
2000
4
f
SO00
0
Control
fra X
I
A
4
0
A
_Control
A
~
~
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.
Discussion In this preliminary study, we provide evidence that some patients with the fragile X syndrome may have
B
A
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
25
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.
References 1, Gilman AG. G proteins: transducers of receptor-generated sig-
nals. Annu Rev Biochem 1987;56:615--649 2. Levirzki A. From epinephrine to cyclic AMP. Science 1988; 24 1:800-806 3. Kandel ER, Schwartz J H . Molecular biology of learning: modulation of transmitter release. Science 1$182;218:433-443 4. D u d i Y. Cyclic AMP and learning in Drosophila. Adv Cyclic Nucleotide Protein Phosphorylation Res 1986;20:343-361 5 . Farfel Z , Brothers VM, Brickman AS, e t al., Pseudohypoparathyroidisni: inheritance of deficient receptor-( yclase coupling activity. I'roc Natl Acad Sci USA 1981;78:30')8-3102 6. Patten JM, Johns DR, Valle D, et al. Muration in the gene encoding the stimulatory G prorein of adenylate cyclase in Albright's hereditary osteodystrophy. N Engl J Med 1990; iLL:1412-14 19 7. Farfel 25, Friedman E. Mental deficiency in pseudohypoparathyroidism type I is associated with N,-protein deficiency. Ann Inrrrn Med 1986;105:197-199 8. Viereg(:e P, Froster-Iskenius V. Cliniccmeurological investigations i n the fra(X) form of mental retardation. J Neurol I089;2 36:85-92
26 Annals of Neurology
Vol 31
No 1 January 1992
9. Reiss AL, Freund L. Fragile X syndrome. Biol Psychiatry 1990; 27:223-240 10. Verkerk AJMH, Pieretti M, Sutcliffe JS, e t al. Ideniification of a gene (I'MK-I) containing a CGG repeat co-incident with a breakpoint cluster region exhihiring length variation in fragile X syndrome. Cell 1991;65:905-914 11. Bell MV, Hirst MC, Nakahori Y . et al. Physical mapping across the fragile X: hypermerhylarion and clinical expression of the fragile X syndrome. Cell 1991;64:861-866 12. Seigl AM, Daly JW, Smith JB. Inhibition of aggregairion and stimulation of cyclic AMP generation by the diterpene forskolin in intact human platelets. Mol Pharmacol 1982;21:680-687 13. Baenziger NL, Majerus PW. Isolation of human platelets and platelet surface membranes. Methods Enzymol 1974;31: 149-1 5 5 14. Motulsky HJ, Hughes RJ, Brickman AS, e t al. Platelets of pseudohypoparathyroid patients: evidence that distinct receptorcyclase coupling proteins mediare stimulation and inhibition ot adenylate cyclase. Proc Natl Acad Sci IJSA 1782;70:41934197 15. Lowry O H , Rosebrough NJ, Farr AL, e t al. Protein measurement with the Folin reagent. J Biol Cheni 1951;193:265275 16. Brooker G, Harper JF, Terasaki WL, et al. Radioimmunoassay of cyclic AMP and cyclic GMP. Adv Cyclic Nucleotide Res 1979;lO: 1-33 17. Seamon KB, Daly JW. Forskolin: its hiological and chemical properties. A& Cyclic Nucleotide Protein Phosphorylation Res 1986;20:1-150 18. Bakalyar HA, Reed RR. Identification of-a specialized adenyl cyclase that may mediate odorant detection. Science 1990; 250:1403-1406 19. Feany MB. Rescue of the learning defect in dunce. a Drosophdu learning mutant, by an allele of rutubugu, a second learning mutant. Proc Natl Acad Sci USA 1990;87:2795-2799 20. Laird CD. Proposed mechanism of inheritance and expression of the human fragile X syndrome of mental retardation. Genetics 1987;117:587-597 21. Pieretti M, Zhang F, Ying-Hui F, et al. Absence o f expression of the FMR-1 gene in fragile X syndromc. Cell 1991;56:817822 22. Webb T. Fragile Xq27 in somatic cells derived from different tissues. Am J Med Gener 1991;38:425-428
~
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,
2)). 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 .
22
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 known.
Materials and Methods Materials 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 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.
*
Results 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
23
Cyciil- AMP Productma in Patients with Fragile X Syndrome and Control Sihjecti
cAMP (prnolirng of protein) Reagents
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 ? 220 145 ? 6,850 i11,300 t-
16
* 30
17 660 1,400
Fra X ( n = 14)
Fra X" (n = 10)
(57)
P
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
87 66 63 61 56
0.651 0.0: 0.019 0.039 0.012
__
-
Fra Xlc
Fra X " l c ('% )
P
77 57
0.49 5 0.053
40
0.0 1 I 0.000 0.032
53 53
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 24
Annals of Neurology
Vol 31 No 1 January 1992
A 14000
R
A
3ouoo
A
12000
25000
loo00
zooom
f 8000
CAMP @mi ms) 6000
A
A A
A
A
A
A CAMP (pmi 15000 mg)
i
f
t
4 A A
A
f
4 10000
A 4
f
4000
'
A
f
2000
4
f
SO00
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fra X
I
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0
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_Control
A
~
~
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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.
Discussion In this preliminary study, we provide evidence that some patients with the fragile X syndrome may have
B
A
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
25
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.
References 1, Gilman AG. G proteins: transducers of receptor-generated sig-
nals. Annu Rev Biochem 1987;56:615--649 2. Levirzki A. From epinephrine to cyclic AMP. Science 1988; 24 1:800-806 3. Kandel ER, Schwartz J H . Molecular biology of learning: modulation of transmitter release. Science 1$182;218:433-443 4. D u d i Y. Cyclic AMP and learning in Drosophila. Adv Cyclic Nucleotide Protein Phosphorylation Res 1986;20:343-361 5 . Farfel Z , Brothers VM, Brickman AS, e t al., Pseudohypoparathyroidisni: inheritance of deficient receptor-( yclase coupling activity. I'roc Natl Acad Sci USA 1981;78:30')8-3102 6. Patten JM, Johns DR, Valle D, et al. Muration in the gene encoding the stimulatory G prorein of adenylate cyclase in Albright's hereditary osteodystrophy. N Engl J Med 1990; iLL:1412-14 19 7. Farfel 25, Friedman E. Mental deficiency in pseudohypoparathyroidism type I is associated with N,-protein deficiency. Ann Inrrrn Med 1986;105:197-199 8. Viereg(:e P, Froster-Iskenius V. Cliniccmeurological investigations i n the fra(X) form of mental retardation. J Neurol I089;2 36:85-92
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9. Reiss AL, Freund L. Fragile X syndrome. Biol Psychiatry 1990; 27:223-240 10. Verkerk AJMH, Pieretti M, Sutcliffe JS, e t al. Ideniification of a gene (I'MK-I) containing a CGG repeat co-incident with a breakpoint cluster region exhihiring length variation in fragile X syndrome. Cell 1991;65:905-914 11. Bell MV, Hirst MC, Nakahori Y . et al. Physical mapping across the fragile X: hypermerhylarion and clinical expression of the fragile X syndrome. Cell 1991;64:861-866 12. Seigl AM, Daly JW, Smith JB. Inhibition of aggregairion and stimulation of cyclic AMP generation by the diterpene forskolin in intact human platelets. Mol Pharmacol 1982;21:680-687 13. Baenziger NL, Majerus PW. Isolation of human platelets and platelet surface membranes. Methods Enzymol 1974;31: 149-1 5 5 14. Motulsky HJ, Hughes RJ, Brickman AS, e t al. Platelets of pseudohypoparathyroid patients: evidence that distinct receptorcyclase coupling proteins mediare stimulation and inhibition ot adenylate cyclase. Proc Natl Acad Sci IJSA 1782;70:41934197 15. Lowry O H , Rosebrough NJ, Farr AL, e t al. Protein measurement with the Folin reagent. J Biol Cheni 1951;193:265275 16. Brooker G, Harper JF, Terasaki WL, et al. Radioimmunoassay of cyclic AMP and cyclic GMP. Adv Cyclic Nucleotide Res 1979;lO: 1-33 17. Seamon KB, Daly JW. Forskolin: its hiological and chemical properties. A& Cyclic Nucleotide Protein Phosphorylation Res 1986;20:1-150 18. Bakalyar HA, Reed RR. Identification of-a specialized adenyl cyclase that may mediate odorant detection. Science 1990; 250:1403-1406 19. Feany MB. Rescue of the learning defect in dunce. a Drosophdu learning mutant, by an allele of rutubugu, a second learning mutant. Proc Natl Acad Sci USA 1990;87:2795-2799 20. Laird CD. Proposed mechanism of inheritance and expression of the human fragile X syndrome of mental retardation. Genetics 1987;117:587-597 21. Pieretti M, Zhang F, Ying-Hui F, et al. Absence o f expression of the FMR-1 gene in fragile X syndromc. Cell 1991;56:817822 22. Webb T. Fragile Xq27 in somatic cells derived from different tissues. Am J Med Gener 1991;38:425-428
