Basile et al (1999) NEUROPSYCHOPHARMACOLOGY. 21 (1): 17

1. Association of the MscI Polymorphism of the Dopamine D3 Receptor Gene with Tardive Dyskinesia in Schizophrenia Basile et al (1999) NEUROPSYCHOPHARMACOLOGY. 21 (1): 17

2. The Nature of Schizophrenia • Effects 1% of the population • One of the most important forms of psychiatric illness as it affects people from an early age • Often chronic and usually highly disabling • Associated with a neurodevelopment disorder affecting mainly the cerebral cortex, and occuring in the first few months of prenatal development. • Strong hereditary factor (twin studies) • Combination of Genetic and Environmental factors (complex trait)

3. Positive Symptoms Delusions ( often paranoid in nature). Hallucinations, usually in the form of voices. Thought disorder, comprising wild trains of thought and irrational conclusions, often associated with the feeling that thoughts are inserted or withdrawn by an outside agency. Clinical features Negative Symptoms Withdrawal from social contacts Flattening of emotional responses

4. Causes Large portion of dopamine content of the brain is found in the corpus striatum, a part of the extrapyramidal motor system concerned with the coordination of movement. • Pharmacological evidence is generally consistent with dopamine over activity hypothesis

5. Dopamine Receptors G-protein-coupled transmembrane receptors D1 family D2 family D2, D3 & D4 D1 & D5

6. Tuberohypophyseal pathway Short neurons running from arcuate nucleus of the hypothalamus to the pituitary gland Hypothalmus Mesolimbic/mesocortical pathway Cell bodies occur in groups in midbrain with fibres projecting, via the medial forebrain bundle, to parts of the limbic system, and to the cortex Corpus striatum Substantia nigra MESOCORTICAL PATHWAY Nigrostriatal pathway 75% dopaminergic neurons ie abundant in the striatum Fibres run in the medial forebrain bundle. Pituitary gland Nucleus accumbens MESOLIMBIC Hippocampus Amygdaloid nucleus Location of the main groups of cell bodies and fibre tracts

7. Drug therapy Although there are no cures, neuroleptic medications are frequently used to alleviate the psychosis. typical neuroleptic drugs = classical antipychotic drugs pre-1980 drugs which are very similar in their properties. Neuroleptic medications = antipychotic drugs

8. Dopamine Antagonist Dopamine produce motor disturbances collectively termed Extra-pyramidal side-effects (EPS) ESP can occur within the first few days or weeks of treatment, or it can appear after months and years of antipsychotic medication use. More than 60% of the people who take conventional antipsychotic medications experience some form of EPS. EPS can cause a variety of symptoms, e.g. involuntary movements, tremors and rigidity, body restlessness, muscle contractions and changes in breathing and heart rate. Accumulation of dopamine metabolites Dopamine synthesis & release D2 Receptor

9. Tardive dyskinesia (TD). Although the mechanism of TD remains unknown, it has been postulated that an over activity of dopaminergic neurotransmission in the basal ganglia may play a crucial role in the manifestation of TD. • motor disorder characterized by abnormal involuntary movements of the orofacial musculature, particularly in the jaw, lips, and tongue. Choreoathetoid movements of the extremities and/or trunk involvement may occur as well. • occurs in predisposed individuals during or following cessation of prolonged typical neuroleptic treatment.

10. Basal Ganglia http://www.driesen.com/basal_ganglia.htm Group of nuclei lying deep in the white matter of the frontal lobes that organizes motor behavior.

11. Corpus striatum Substantia nigra The dopamine overactivity hypothesis for TD dopamine agonists increase TD severity, whereas, dopamine antagonists reduce the symptoms It has been suggested that the neuroleptic-induced increase in dopamine receptor density eventually results in an overactivity of the nigrostriatal dopaminergic tract TD results from chronic neuroleptic blockade of dopamine D2-like receptors in the basal ganglia long-term blockade results in an upregulation of dopamine receptors on postsynaptic membranes

12. 20–30% of patients suffer from neuroleptic-induced TD - implying variable susceptibility susceptibility and/or protection, predisposing only certain patients to TD Evidence form animal models Example Cebus monkey, even after several years of neuroleptic administration, only some develop this disorder

13. Genetic Predisposition Studies - family studies localized dopamine D3 mRNA and protein to the ventral side of the striatum and the ventral putamen in the basal ganglia. Candidate Genes human postmortem study illustrated a 45 to 56% increase in the number of D3 receptors in the basal ganglia of neuroleptic-treated schizophrenics - initially D2 receptor gene was thought to be the most likely candidate. study revealed a twofold increase in the number of D3 receptors in the basal ganglia of long-term hospitalized patients with schizophrenia. dopamine D3 receptors provide an inhibitory effect on locomotor activity. - Dopamine D3 receptor

14. dopamine receptor D3 (DRD3) 3q13.3 This gene encodes the D3 subtype of the dopamine receptor. The D3 subtype inhibits adenylyl cyclase through inhibitory G-proteins. This receptor is expressed in phylogenetically older regions of the brain, suggesting that this receptor plays a role in cognitive and emotional functions. It is a target for drugs which treat schizophrenia and Parkinson disease. Alternative splicing of this gene results in five transcript variants encoding different isoforms. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retrieve&dopt=Graphics&list_uids=1814

15. Predisposing Polymorphism The Ser9Gly polymorphism denotes a serine to glycine amino acid substitution in the N-terminal extracellular domain of the D3 receptor additionally allelic differences in affinity for dopamine have been shown

16. Table 1. Demographic Distributions of the Sample

17. Patients received at least three periods of treatment in the preceding 5 years at doses equivalent to or greater than 1000 mg/d of chlorpromazine for a period of at least 6 weeks. • None of the patients had ever been treated with atypical neuroleptics. • Patients underwent a washout period of 2–4 weeks which allows the TD phenotype to be expressed more fully if they carry a predisposing factor. • TD severity measured using either the Abnormal Involuntary Movement Scale (AIMS) or a modified Hillside Simpson Dyskinesia scale. • Genotyping

18. Table 1. Demographic Distributions of the Sample

19. Interesting Observations • African Americans in the sample had higher mean AIMS scores than the other patients of different ethnic origins • Significant linear relationship was found between age and TD severity. More specifically, as age increased, patients were more likely to exhibit higher AIMS scores • Statistically significant difference between genotype frequency and ethnic status. African Americans had a significantly higher occurrence of the glycine allele of the Ser9Gly polymorphism than did the Caucasians.

20. serine to glycine polymorphism in the DRD3 receptor was significantly associated with typical neuroleptic-induced TD. • higher mean AIMS scores in patients homozygous for the glycine allele of the DRD3 gene when compared to heterozygous patients and patients homozygous for the serine allele. Figure 1. Mean corrected AIMS scores for each of the genotypic classes. The variances(s2) for each of the genotypic classes were as follows: (s2 for Ser/Ser genotype was 18.8; s2 for Ser/Gly genotype was 28.4; and s2 for Gly/Gly genotype was 156.1). The Levene Test for homogeneity of variances revealed a significant difference in the variances among the genotypic classes, thus violating one of the assumptions of the ANCOVA model. However, a nonparametric alternative, the Kruskal–Wallis Test, illustrated similar results ( 2 = 13.6644, df = 2, p = .0011, p = 0.0033 Bonferroni corrected)

21. To test for effects of ethnic stratification, we conducted an analysis that separated Caucasians and African Americans. It was found that in both Caucasians (n = 85, F[2,75] = 3.85, p = .026) and African Americans (n = 25, F[1,23] = 8.10, p = .0091), patients homozygous for the glycine allele were more likely to exhibit higher AIMS scores. Figure 2. Ethnically stratified analysis. To address the issue of population stratification, an analysis separating Caucasians from African Americans was conducted. It was found that in both ethnic samples, patients with Gly/Gly genotypes were more likely to exhibit larger AIMS scores, thus reducing the possibility of a false positive attributable to ethnic stratification. Figure 2A illustrates the Caucasian results, and Figure 2B gives the African American results

22. Conclusion • observed a statistically significant association between the glycine variant of DRD3 and TD. • mean AIMS scores for individuals homozygous for the glycine variant were significantly higher than individuals who were either serine/serine homozygotes or serine/glycine heterozygotes.

23. Parallels The substitution of a polar serine residue to a nonpolar glycine residue may alter the tertiary structure of the D3 receptor. The functional differences in dopamine affinity for each of these receptor alleles as expressed in CHO cell lines have been identified There is a higher dopamine affinity for the glycine homozygote cells, as compared to both heterozygote binding and serine/serine homozygotesin these cells.

24. Problems • Factors examined D3 genotype age sex ethnicity D3 genotype was the only factor that significantly contributed to the TD phenotype. environmental influences ? total neuroleptic histories for all patients were unavailable phenotypic heterogeneity

25. Closing Remarks • As the data in the study correlates to the CHO cell study findings perhaps the functional differences may contribute to the TD phenotype, although it is difficult to speculate further on the precise mechanism involved. • It could be suggested that the higher affinity of the glycine allele may be involved in the pathogenesis of TD. Alternatively, this Ser9Gly polymorphism may be in linkage disequilibrium with another site conferring susceptibility to TD. • The results presented in this study suggest the involvement of the Ser9Gly DRD3 polymorphism as a predisposing risk factor for TD. This genetic information may, in future, assist clinicians in determining patient susceptibility to TD and ultimately may aid in the elucidation of the pathophysiological mechanisms.