An association of NAG levels and a mutation of the CCK gene in panic disorder patients

Psychiatry Research 80 Ž1998. 149]153

An association of NAG levels and a mutation of the CCK gene in panic disorder patients Michael J. Garvey a,b,U , Raymond R. Croweb , Zhewu Wang b a

b

Department of Psychiatry, VA Medical Center, Iowa City, IA 52246-2208, USA Department of Psychiatry, Uni¨ ersity of Iowa College of Medicine, Iowa City, IA 52246, USA

Received 9 February 1998; received in revised form 18 May 1998; accepted 29 May 1998

Abstract Levels of the enzyme N-acetyl-b-glucosaminidase ŽNAG. and a mutation of cholecystokinin ŽCCK. gene appear to be independently associated with panic disorder. We explored whether there was an association of NAG levels and a CCK mutation identified in a group of panic disorder patients. NAG was measured in 12 panic disorder patients who had a mutation of the CCK gene and 17 who did not. Urine for NAG was collected at baseline and after 3 and 6 weeks of treatment. NAG levels were lower at all three times in the patients that did not have the CCK mutation. The difference between the two groups was significant at week 6 Ž P- 0.02., and showed a trend toward a difference at baseline Ž P- 0.15.. Q 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: N-acetyl-b-glucosaminidase; Cholecystokinin-4; Genetics; Panic disorder

1. Introduction Cholecystokinin ŽCCK., a peptide, is a CNS neurotransmitter. There are various active forms of CCK. The octapeptide CCK-8 is the most abundant. The tetrapeptide, CCK-4, induces dose-dependent panic attacks in some healthy

U

Corresponding author. Tel.: q1 319 3397176; fax: q1 319 3397095.

control subjects and in most patients with panic disorder ŽBradwejn et al., 1991a,b, 1992a,b; Bradwejn and Koszycki, 1994; van Megen et al., 1996.. Benzodiazepines decrease the behavioral effects of CCK-4 in control subjects Žde Montigny, 1989.. Fluvoxamine and imipramine, are effective treatments for panic disorder. Both antagonize the panicogenic effects of CCK-4 ŽBradwejn and Koszycki, 1994; van Megen et al., 1997.. Panic patients had lower concentrations of cerebrospinal fluid CCK-8 compared to healthy con-

0165-1781r98r$19.00 Q 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0165-1781Ž98.00063-8

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trol subjects ŽLydiard et al., 1992.. These data suggest that CCK andror the CCK B receptor may play a role in panic disorder. A recent analysis of the CCK gene revealed a point mutation of the promoter region in 33% of 48 patients with panic disorder ŽWang et al., 1998.. This was significantly different from the 16% frequency found in 247 geographically matched control subjects. Twenty-nine of these 48 panic disorder patients also provided urine samples that were analyzed for the enzyme N-acetylb-glucosaminidase ŽNAG.. We have previously shown that NAG levels in patients with panic disorder were significantly lower than those in healthy control subjects ŽGarvey et al., 1993.. Significant associations were also reported between urinary NAG and the severity of various psychiatric symptoms in a group of panic disorder patients ŽGarvey and Noyes, 1996.. Both NAG and a mutation of CCK appear to be associated with panic disorder. We explored whether there was an association of NAG levels and the CCK mutation identified in these panic disorder patients. 2. Methods Patients were solicited through advertising for a treatment study of panic disorder ŽBallenger et al., 1988.. Patients were screened with the Structured Clinical Interview for DSM-III ŽSCID, Upjohn version. ŽSpitzer and Williams, 1982.. Those meeting DSM-III criteria for panic disorder were invited to participate. Patients were asked about various features of their illness including demographics, longitudinal course, and comorbidity. Consenting patients were invited to collect urine samples for cortisol ŽKathol et al., 1988.. The majority of patients were not taking psychotropic medications when they sought treatment. Those that were stopped taking their medications at least 1 week before collecting urine samples. In previous studies we have not found any relationship between use of a variety of psychotropic medications and NAG levels ŽGarvey et al., 1982, 1988.. Some nephrotoxic medications and certain diseases can cause NAG elevations ŽWollen and Turner, 1965; Burry et al., 1976;

Kunin et al., 1978; Mansell et al., 1978; Dubach and Schmidt, 1979; Maruhn, 1979; Whiting et al., 1979; Reglero et al., 1980; Tucker et al., 1980; Price, 1982; Alderman et al., 1983.. No patients were using nephrotoxic medications. Patients with serious medical illness were excluded from the study. If any inappropriate patients were inadvertently included, it should have decreased our ability to find significant relationships between NAG and the CCK mutation. Urine collection procedures have been detailed elsewhere ŽKathol et al., 1988.. Briefly, 24-h samples were collected and frozen at y208C. NAG was analyzed approx. 2]3 years after the collections. We have found NAG levels to remain stable for more than 4 years when frozen at y208C Žunpublished data.. Urine samples were analyzed for NAG by a method described by Price et al. Ž1970.. Aliquots of urine were mixed with buffered solution containing 4-methylumbelliferyl-N-acetyl-b-D-glucosaminide for a predetermined time. NAG catalyzed the release of the fluorescent compound 4-methylumbelliferyl. The NAG enzyme activity was calculated from the quantity of released substrate which was measured by a fluorometer ŽPrice et al., 1970; Tucker et al., 1975; Wellwood et al., 1976.. To adjust for variations in urine flow, the enzyme activity is divided by the concentration of creatinine ŽWellwood et al., 1976.. The final NAG value is reported in nanomoles of released substrate per hour per milligram of creatinine. Previous studies have demonstrated that the NAG assay is simple, reliable, and has a coefficient of variation for duplicate samples of approx. 2% ŽTucker et al., 1975; Wellwood et al., 1976.. Several years after the treatment study was completed ŽBallenger et al., 1988., patients that could be located were invited to participate in a study of CCK gene analysis. Details of the CCK analysis have been reported elsewhere in detail ŽWang et al., 1998.. Briefly, DNA was collected from white blood cells. The promoter region and three exons were amplified with four sets of PCR Žpolymerase chain reaction. primers. Purified DNA was sequenced by the cycle sequence method using an Amplicycle sequencing kit ŽPerkin-Elmer..

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NAG levels were determined at baseline and after 3 and 6 weeks of treatment ŽBallenger et al., 1988.. A Student’s t-test was used to compare NAG levels for patients with and without the CCK mutation for each of the three time points. 3. Results Ninety patients participated in the panic treatment study ŽBallenger et al., 1988.. Forty-eight were located several years after the treatment study and consented to gene analysis. Twenty-nine of these 48 patients collected urine samples at the time of the treatment study. These 29 patients are the subject of this report. These patients had mean Ž"S.D.. age of 39 " 8 years. Forty-eight percent of the sample were women. The mean Ž"S.D.. Hamilton Anxiety Rating Scale score was 21 " 9. The baseline urinary NAG level was 14 " 10. ŽFor reference a mean NAG level for 38 healthy control subjects was 27 " 14; Garvey et al., 1990.. Not all patients completed the 6-week study. Therefore data were available for 26 and 18 patients at 3 and 6 weeks, respectively. NAG levels for patients that had the CCK mutation were consistently greater than those of patients with no mutation at all three comparison times ŽTable 1.. At baseline this difference showed a statistical trend that did not reach significance in a two-tailed Student t-test Ž t s 1.5; Ps 0.15; d.f.s 27.. The comparison made at week 6 was significant Ž t s 2.6; Ps 0.02; d.f.s 16.. 4. Discussion Patients that did not have the CCK mutation had consistently lower levels of NAG at all three sampling times ŽTable 1.. What this finding might mean is open to speculation. There is some indirect evidence that NAG may be a marker for serotonin. If true, then the low NAG levels in panic disorder patients might suggest that these patients have reduced serotonin metabolism. There is support for a hypothesis of low serotonin in anxiety disorders ŽCharney et al., 1987; Kahn et al., 1988; Charney et al., 1990; Kahn et al., 1991.. Low serotonin could play a

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Table 1 NAG levels in patients with and without a CCK mutation NAG ŽMean " S.D..

CCK mutation patients Ž N s 12. ‘Normal’ CCK patients Ž N s 17. U

Baseline

Week 3

Week 6

U

17 " 13

15 " 12

26 " 17UU

12 " 6U

12 " 7

11 " 7UU

Ps 0.15, two tailed Student t-test. Ps 0.02, two tailed Student t-test.

UU

causal or permissive role in anxiety. In panic patients with the lowest NAG levels, an abnormality of serotonin metabolism may play a pivotal role in their disorder. For anxiety patients with NAG levels nearer control values, factors besides serotonin metabolism may be of importance. An example of such a factor could be release of CCK. Studies have shown that CCK can induce panic attacks in a dose-dependent fashion ŽBradwejn et al., 1991a,b, 1992a.. There are several studies that show a relationship between CCK and serotonin in the central nervous system ŽPinnock et al., 1990; Raiteri et al., 1993; Vasar et al., 1993; Bickerdike et al., 1994; Rex et al., 1994, 1997.. The mutation in the CCK gene was a C to T substitution at positiony 36 in the GC box of the promoter region ŽWang et al., 1998.. Most often such substitutions would lead to the promoter region not working as effectively and resulting in decreased production of the corresponding mRNA and its peptide product, in this case CCK. If this reasoning is correct then patients with this mutation could have an erratic output of CCK. Conceivably such an output could lead to panic symptoms. If this later speculation were true it would be consistent with the finding that panic patients with the CCK mutation had NAG levels Žand by proxy serotonin levels. that were closer to those found in healthy control subjects. In other words their NAG Žserotonin. was not markedly reduced and another factor, namely erratic release of CCK, was of importance in the production of panic symptoms.

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The empirical finding of an association between low NAG and panic patients’ not having the CCK mutation could be explained by a variety of other hypotheses. It is important to recognize that this is only a preliminary study that requires replication. There are no substantive data to support any explanatory hypotheses. Both NAG levels and a CCK mutation have been found to be associated with panic disorder. The association we found between NAG and a CCK mutation in panic disorder patients should be of some value for further hypothesis generation and testing. Acknowledgements Support for this study was provided by the Department of Veterans Affairs, the University of Iowa College of Medicine, and National Institute of Mental Health research grants R01MH34728 and K02MH00735. References Alderman, M.H., Melcher, L., Drayer, D.E., Reidenberg, M.M., 1983. Increased excretion of N-acetyl-beta-glucosaminidase in essential hypertension and its decline with antihypertensive therapy. New England Journal of Medicine 309, 1213]1217. Ballenger, J.C., Burrows, G.D., Du Pont, R.L., Jr., Lesser, I.M., Noyes, R., Jr., Pecknold, J.C., Rifkin, A., Swinson, R.P., 1988. Alprazolam in panic disorder and agoraphobia: results from a multicenter trial. I. Efficacy in short-term treatment. Archives of General Psychiatry 45, 413]422. Bickerdike, M.J., Marsden, C.A., Dourish, C.T., Fletcher, A., 1994. The influence of 5-hydroxytryptamine re-uptake blockade on CCK receptor antagonist effects in the rat elevated zero-maze. European Journal of Pharmacology 271, 403]411. Bradwejn, J., Koszycki, D., 1994. Imipramine antagonism of the panicogenic effects of cholecystokinin tetrapeptide in panic disorder patients. American Journal of Psychiatry 151, 261]263. Bradwejn, J., Koszycki, D., Annable, L., Couetoux du Tertre, A., Reines, S., Karkanias, C., 1992a. A dose-ranging study of the behavioral and cardiovascular effects of CCK-tetrapeptide in panic disorder. Biological Psychiatry 32, 903]912. Bradwejn, J., Koszycki, D., Bourin, M., 1991a. Dose ranging study of the effects of cholecystokinin in healthy volunteers. Journal of Psychiatry and Neuroscience 16, 91]95. Bradwejn, J., Koszycki, D., Payeur, R., Bourin, M., Borthwick, H., 1992b. Replication of action of cholecystokinin tetrapeptide in panic disorder: clinical and behavioral findings. American Journal of Psychiatry 149, 962]964.

Bradwejn, J., Koszycki, D., Shriqui, C., 1991b. Enhanced sensitivity to cholecystokinin tetrapeptide in panic disorder. Clinical and behavioral findings. Archives of General Psychiatry 48, 603]610. Burry, H.C., Dieppe, P.A., Bresnihan, F.B., Brown, C., 1976. Salicylates and renal function in rheumatoid arthritis. British Medical Journal 1, 613]615. Charney, D.S., Woods, S.W., Goodman, W.K., Heninger, G.R., 1987. Serotonin function in anxiety: II. Effects of the serotonin agonist mCPP in panic disorder patients and healthy controls. Psychopharmacology 92, 14]24. Charney, D.S., Woods, S.W., Krystal, J.H., Nagy, L.M., Heninger, G.R., 1990. Hypothesis relating serotonergic dysfunction to the etiology and treatment of panic and generalized anxiety disorders. In: Coccaro, E.F., Murphy, D.L. ŽEds.., Serotonin in Major Psychiatric Disorders. Progress in Psychiatry No. 21. American Psychiatric Press, Washington, DC, pp. 129]152. de Montigny, C., 1989. Cholecystokinin tetrapeptide induces panic-like attacks in healthy volunteers. Preliminary findings. Archives of General Psychiatry 46, 511]517. Dubach, A.C., Schmidt, A., 1979. Diagnostic Significance of Enzymes and Protein in Urine. Hans Huber Publishers, Bern, Stuttgart, Vienna. Garvey, M.J., Furlong, C., Schaffer, C.B., Adelman, R., 1988. Elevation of the renal enzyme N-acetyl-beta-glucosaminidase in affective disorders and chemical dependence. Acta Psychiatrica Scandinavica 77, 247]252. Garvey, M.J., Noyes, R., Jr., 1996. Association of levels of N-acetyl-beta-glucosaminidase with severity of psychiatric symptoms in panic disorder. Psychiatry Research 60, 185]190. Garvey, M., Noyes, R.J., Cook, B., Barrickman, L., Noel, M., Ghosheh, R., 1990. Elevated levels of N-acetyl-beta-glucosaminidase in affective disorders and chemical dependence. Journal of Affective Disorders 19, 279]285. Garvey, M.J., Noyes, R.J., Woodman, C., Laukes, C., 1993. A biological difference between panic disorder and general anxiety disorder. Biological Psychiatry 34, 572]575. Garvey, M.J., Tuason, V.B., Blomquist, C.H., Hwang, S., 1982. Use of renal enzymes to evaluate nephrotoxicity in lithium treated patients. British Journal of Psychiatry 141, 420]422. Kahn, R.S., Kalus, O., Wetzler, S., van Praag, H., 1991. The Role of Serotonin in the Regulation of Anxiety. BrunnerrMazel, New York. Kahn, R.S., Wetzler, S., van Praag, H., Asnis, G.M., Strauman, T., 1988. Behavioral indications for serotonin receptor hypersensitivity in panic disorder. Psychiatry Research 25, 101]104. Kathol, R.G., Noyes, R.J., Lopez, A.L., Reich, J., 1988. Relationship of urinary free cortisol levels in patients with panic disorder to symptoms of depression and agoraphobia. Psychiatry Research 24, 211]221. Kunin, C.M., Chesney, R.W., Craig, W.A., England, A.C., DeAngelis, C., 1978. Enzymuria as a marker of renal injury and disease: studies of N-acetyl-beta-glucosaminidase in the general population. Pediatrics 62, 751]760.

M.J. Gar¨ ey et al. r Psychiatry Research 80 (1998) 149]153 Lydiard, R.B., Ballenger, J.C., Laraia, M.T., Fossey, M.D., Beinfeld, M.C., 1992. CSF cholecystokinin concentrations in patients with panic disorder and in normal comparison subjects. American Journal of Psychiatry 149, 691]693. Mansell, M.A., Jones, N.F., Ziroyannis, P.N., Marson, W.S., 1978. N-acetyl-beta-glucosaminidase: a new approach to screening hypertensive patients for renal disease. Lancet 2, 803]805. Maruhn, D., 1979. Evaluation of urinary enzyme patterns in patients with kidney disease and primary benign hypertension. Current Problems in Clinical Biochemistry 9, 135]149. Pinnock, R.D., Woodruff, G.N., Boden, P.R., 1990. Cholecystokinin excites dorsal raphe neurons via CCK A receptors. British Journal of Clinical Pharmacology 100 suppl, p. 349. Price, R.G., 1982. Urinary enzymes, nephrotoxicity, and renal disease. Toxicology 23, 99]135. Price, R.G., Dance, N., Richard, B., Catell, W.R., 1970. The excretion of N-acetyl-beta-glucosaminidase and betagalactosidase following surgery to the kidney. Clinica Chimica Acta 27, 65]72. Raiteri, M., Paudice, P., Vallebuona, F., 1993. Release of cholecystokinin in the central nervous system wsee commentsx. Neurochemistry International 22, 519]527. Reglero, A., Carretero, I., Cabezas, J.A., 1980. Increased serum alpha-L-fuctosidase and beta-N-acetylglucosaminidase activities in diabetic, cirrhotic, and gastric cancer patients. Clinica Chimica Acta 103, 155]158. Rex, A., Fink, H., Marsden, C.A., 1994. Effects of BOC-CCK-4 and L 365.260 on cortical 5-HT release in guinea-pigs on exposure to the elevated plus maze. Neuropharmacology 33, 559]565. Rex, A., Marsden, C.A., Fink, H., 1997. Cortical 5-HT-CCK interactions and anxiety-related behaviour of guinea-pigs: a microdialysis study. Neuroscience Letters 228, 79]82. Spitzer, R.L., Williams, B.W., 1982. Structured Clinical Interview for DSM-III. Biometrics Research Department, New York State Psychiatric Institute, New York.

153

Tucker, S.M., Boyd, P., Thompson, A.E., Price, R.G., 1975. Automated assay of N-acetyl-beta-glucosaminidase in a normal pathological human urine. Clinica Chimica Acta 62, 333]339. Tucker, S.M., Pierce, R.J., Price, R.G., 1980. Characterization of human N-acetyl-b-glucosaminidase isoenzymes as an indicator of tissue damage in disease. Clinica Chimica Acta 102, 29]40. van Megen, H.J., Westenberg, H.G., den Boer, J.A., Kahn, R.S., 1996. Cholecystokinin in anxiety. European Neuropsychopharmacology 6, 263]280. van Megen, H.J., Westenberg, H.G., den Boer, J.A., Slaap, B., Scheepmakers, A., 1997. Effect of the selective serotonin reuptake inhibitor fluvoxamine on CCK-4 induced panic attacks. Psychopharmacology 129, 357]364. Vasar, E., Peuranen, E., Oopik, T., Harro, J., Mannisto, P.T., 1993. Ondansetron, an antagonist of 5-HT3 receptors, antagonizes the anti-exploratory effect of caerulein, an agonist of CCK receptors, in the elevated plus-maze. Psychopharmacology 110, 213]218. Wang, Z., Valdes, J., Noyes, R., Zoega, T., Crowe, R.R., 1998. Possible association of a cholecystokinin promoter polymorphism ŽCCK-36C-)T. with panic disorder. American Journal of Medical Genetics 81, 228]234. Wellwood, J.M., Price, R.G., Ellis, B.G., Thompson, A.E., 1976. A note on the practical aspects of the N-acetyl-betaglucosaminidase in human urine. Clinica Chimica Acta 69, 85]91. Whiting, P.H., Ross, I.S., Borthwick, L., 1979. Serum and urine N-acetyl-beta-glucosaminidase in diabetics on diagnosis and subsequent treatment, and stable insulin dependent diabetics. Clinica Chimica Acta 92, 459]463. Wollen, J.W., Turner, P., 1965. Plasma N-acteyl-beta-glucosaminidase and beta-glucuronidase in health and disease. Clinica Chimica Acta 12, 671]683.