American Journal of Medical Genetics (Neuropsychiatric Genetics) zy 60:452455 (1995) z t zyxwvutsrq Association Study Between the Dopamine D4 Receptor Gene and Schizophrenia zy ~~ ~~~~~ Arturas Petronis, Fabio Macciardi, Andrew Athanassiades, Andrew D. Paterson, Massimiliano Verga, Herbert Y. Meltzer, Philip Cola, Janet A. Buchanan, Hubert H.M. Van Tol, and James L. Kennedy zyxwvuts Department of Neurogenetics (A.P., F.M., A.A., A.D.P., J.L.K.) and Molecular Psychobiology Laboratory (H.H.M.V.T.), Clarke Institute of Psychiatry, and Molecular Genetics Laboratory (J.A.B.), North York General Hospital, Toronto, Ontario, Canada; Department of Neuroscience (F.M., M.V.), Istituto Scientific0 H San Raffaele, University of Milan, Milan, Italy; and Department of Psychiatry (H.Y.M., P.C.), Case Western Research University, Cleveland, Ohio The dopamine D4 receptor is of major inter- est in schizophrenia research due to its high affinity for the atypical neuroleptic cloza- pine and a high degree of variability in the receptor gene (DRD4).Although several ge- netic linkage analyses performed on schizo- phrenia multiplex families from different regions of the world have either excluded or failed to prove that DRD4 is a major genetic factor for the development of schizophre- nia, analyses for moderate predisposing effects are still of significant interest. We performed a study examining differences in allele frequencies of 4 different DRD4 poly- morphisms in schizophrenia patients and age, sex, and ethnic origin matched controls. None of these 4 polymorphisms showed evi- dence for genetic association with schizo- phrenia, although a trend towards excess of the allele with 7 repeats in the (48), bp exon I11 polymorphism was observed. Complexi- ties in the DRD4 genetic investigation and further analytic approaches are discussed. zyxwv 0 1995 Wiley-Liss, Inc. KEY WORDS: DNA polymorphism, neuro- leptic clozapine, association study, dopamine D4 receptor gene INTRODUCTION The dopamine hypothesis of schizophrenia is one of the strongest to be investigated by schizophrenia re- searchers [reviewed by Seeman, 19871. The dopamine Received for publication June 9, 1994; revision received August zyxwvuts 25, 1994. Address reprint requests to James L. Kennedy, Department of Neurogenetics, Clarke Institute of Psychiatry, 250 College Street, Toronto, Ontario M5T 1R8, Canada. 0 1995 Wiley-Liss, Inc. D4 receptor [DRD4; Van To1 et al., 19911 has recently attracted major interest in schizophrenia research for several reasons. D4 demonstrates significantly higher affinity, in comparison to the D2 dopamine receptor, for the atypical antipsychotic clozapine [Van To1 et al., 19911. A highly variable 48 base pair (bp) repeat poly- morphism has been discovered in exon I11 of the DRD4 gene [Van To1 et al., 19921. It was demonstrated that the dissociation constant for clozapine, in the absence of sodium, differs between variants of this polymor- phism and thus has differences with respect to ligand binding [Van To1 et al., 19921. Subtraction of the maxi- mum receptor density by differential binding of ligands revealed the amount of D4-like sites in the postmortem brain of schizophrenic patients was increased 6-fold as compared to control tissues [Seeman et al., 19931. Unfortunately, despite the strong biochemical and pharmacological evidence, several linkage studies on schizophrenia multiplex families have shown evidence excluding DRD4 as a major gene causing schizophrenia under the assumed models of transmission [Kennedy et al., 1993; Barr et al., 1993; Shaikh et al. 1994; Macciardi et al., 19941. Association studies of DRD4 al- leles and schizophrenia were performed by several groups [Cichon et al., 1993; Shaikh et al., 1993; Yang et al., 19931 and no statistically significant differences were noted in allele frequencies between patients and controls. Recently, a new polymorphism was detected in the first exon of DRD4 by Catalano et al., [1993]. This poly- morphism consists of a 12 bp DNA fragment which occurs as a 2-fold repeat in the most common variant and is represented only once in in the rarer variant. Ini- tial reports suggest that this polymorphism may be a risk factor in delusional disorders and possibly obses- sive-compulsive disorders but not in schizophrenia [Catalano et al., 19931. Based on the above rationale, we have performed an extended association study of 4 polymorphisms of the DRD4 gene with schizophrenia. In addition to the 48 bp repeat in exon I11 and the 12 bp deletion in exon I, we detected and analyzed 2 new polymorphisms. Both af-