Association Between A Ser-9-Gly Polymorphism In The Dopamine D3 Receptor Gene And Schizophrenia

PrefaceSchizophrenia is a common disorder with a lifetime prevalence of approximately 1%. It is highly heritable but the genetics are complex. Abnormal dopaminergic transmission has been suggested to play a central role in the pathogenesis of schizophrenia. Most neuroleptics used in clinical pratice bind with the D3 receptor . Expression of the DRD3 gene localized to limbic areas of the brain, which are associated with cognitive, emotional, and endocrine functions.Therefore, the dopamine D(3) receptor gene ( DRD3) ,mapped to chromosome 3q13. 3, became a candidate for a number of psychiatric conditions including schizophrenia. The DRD3 gene exhibits a single - nucleotide (SNP) polymorphism which is named Ser9Gly polymorphism in the first exon that results in a serine - to - glycine amino acid substitution (Ser9Gly ) in the N - terminal extracellular domain of the D3 receptor. Initial genetic case control studies suggested an association between the Ser9Gly variant and schizophrenia. Subsequent studies yielded variable results. Associating the clinical matierl for age,sex, a-ges at onset, and psychiatric symptoms , We conducted a case - control association study in Han Chinese schizophrenic patients and healthy matched controls from Shenyang to detecte the genotype distribution of northern area and examine the relationship of this serine - to - glycine polymorphism and schizophrenia.Materials1. samplePatient sample: A total of 70 schizophrenic patients of han origin who fulfilled DSM - Ⅳ criteria for schizophrenia were recruited . Patient group consisted of 33 males and 37 females, mean age 35.04 ±8.33yearsControl sample:94 ethnically matched controls were obtained from the Shenyang blood donor service. The control group consisted of 46 males and 55 females, with mean age of 35.04 ±8.33years.The x2 test was perform to significance of sex. The t - test was usedto compare ages between cases and controls. There were no significant between - group differences in sex or age. ( sex:x2 = 0. 02, P > 0. 05, age: t = 1. 393, P > 0. 05).2. experimental reagent: Proteinase K, Taq DNA polymerase, Primer , DNTP,PCR buffer,Restriction Enzyme : MscI3. Instrument: High Speed Refrigerated Centrifuge, Constant Temperature Baths, Gene Amp PCR Sysrem, Horizontal Gel Electrophoresis System, Electrophoresis Gel Imaging SystemMethods1. DNA ExtractionWhole blood was collected in disodium EDTA containers Genomic DNA was extracted from the subjects peripheral leucocytes blood according to salting out procedure .2. The polymorphic region of DRD3 amplified by polymerase chain reaction (PCR).forward primer 5' GCT CTATCT CCA ACT CTC ACA 3' reverse primer 5 AAG TCT ACT CAC CTC CAG GTA 3' PCR was performed in a 30 μL total reaction volume containing 5. Oul genomic DNA , each primer(20UM)0. 6ul, 10 × PCR buffer 3 ul, dNTP 2.4ul, Taq DNA polymerase 0.16ul(0.5U)Samples were preheated to 95℃ for 5 min followed by 35cycles of denatur-ation at 94℃ for 30 s, annealing at 56℃ for 45 s and extension at 72℃ for1 min, with a final extension step at 72℃ for 10 min.The PCR products electrophoresis in a 1.5% agarose gel and visualized u-sing the ethidium bromide.3. 20 μL aliquot was incubated overnight with 2 U of MscI restriction . Fragments were separated in a 2.5% agarose gel and visualized by ethidium bromide staining.4. StatisticsStatistical analyses were conducted using the packages SPSS to compared the allele and genotype frequencies among cases and controls.Result1. The distribution of the DRD3 genotypes was in Hardy - Weinberg equilibrium for patients and controls.2. The Ser9Gly polymorphism distribution between patients and controls. There were no significant differences of genotype counts ( x2 =0. 389 P = 0. 823) or allele frequencies (x2 =0.326 P= 0.568) between patients and controls. A small increase in the frequency of the genotype Gly/Gly was observed, however this differences wasn' t statistically significant . The Ser/Ser was most common in two groups. Frequencies of allele Gly in patient were more than that of control group. The allele Ser was most common in two groups.3. Assosiation between Ser9Gly polymorphism and age of onset . The patient were divided to two group according to mean onset age. The frequencies of thoese with onset age before 25 years old were more than that of thoese with onset age after 25 years old but the difference wasnt significant.4. Control groups of different geographical area Ser9Gly polymorphism comparion . Studies were clustered with regard to geographical area . I performed a new analysis on each of these seven ethnic groups . No significant differences of genotype distribution among our study control samples and control samples of Japan , USA, Spain; France and Germany, but there were significant differences of genotype distribution among our study control samples and control samples of Brazil, UK, ( x2 -12.583,12.253respectively, P = 0.02,0.002 respectively).DiscussionAlterations in dopamine transmission and dopamine receptors have since long been hypothesized in the pathophysiology of schizophrenia. The dopamine D3 receptor was found and cloned until 1990. It is localized in limbic areas of the brain which are associated with cognitive, emotional functions. Both typical and atypical antipsychotics block D3 receptors. Recent evidence suggests that a-typical antipsychotics show high affinity to prefrontal D3 receptors. DRD3 - deficient mice are displayed increased locomotor activity and an effect not associated with anxiety state.DRD3 mRNA is predominantly expressed in limbic areasof the brain. Expression of D3 receptor mRNA in schizophrenic brain is very special. Postmortem study of schizophrenic brain reported that the parietal and motor cortices of schizophrenic patients exhibited a selective loss of expression of D3 receptor mRNA. Another postmortem study in patients with schizophrenia who were previously treated with typical antipsychotics illustrated a 45 - 56% increase in the number of D3 receptors in the basal ganglia as compared with controls. Talllan et demonstrated a correlation between the D3 dopamine receptor on lymphocytes and schizophrenia and showed a significant elevation of at least 2 - fold in the mRNA level of the D3 dopamine receptor in schizophrenic patients. The increase was not affected by different antipsychotic drug treatments. Nonmedicated patients exhibited the same pattern, indicating that this change was not a result of medical treatment. It has been suggested that these dopamine receptors found on lymphocytes reflect the receptors found in the brain.Therefore, the dopamine receptor genes are promising candidates for genetic association studies. The DRD3 gene exhibits ser9 - to - gly polymorphism. A functional study of this Ser9Gly polymorphism in CHO cells revealed allelic differences in affinity for dopamine. Specifically, a significantly higher affinity for dopamine was shown for cells homozygous for glycine than heterozygotes and serine/serine homozygotes. It is likely that the substitution of a polar serine residue by a non - polar glycine residue might have altered the tertiary structure ofthe receptor, thus affecting its binding affinity for dopamine.Initial genetic case control studies suggested an association with homozygosity at this locus Crocq et al. (1992) , Subsequent studies yielded variable results: In Chinese, the results show that there may exist association between DRD3 and schizophrenia , also schizophrenia siblings with Sei9 allele are apt to have negative symptoms. Tang et al found significant difference in genotypic or allelic distributions ofDRD3 Ser9Gly polymorphism between patients and controls.We have provieded the distribution of Ser9Gly either allele frequencies or genotype distribution. The main genetype of this area Han people is Ser/Ser and allele Ser is 72. 9%. The main allele in schizophrenia is Ser too (70% ). No significant differences in genotypes and alleles frequencies of Ser 9 Gly polymorphism in DRD3 were observed between schizophrenic patients and controls. When patients were divided according to age at onset, the differences were still not significant.Association studies of Ser9Gly polymorphism and schizophrenia have produced conflicting findings. It may have several explanations. First, schizophreni-a is a heterogenous disorder, in which genetic factors may have a limitedrole. Second, the phenotypic heterogeneity of the disorder (e.g. , different syndromic clusters, variation in familiality, age at onset, drug responsiveness, or comorbid disorder) renders the recruitment of rigourously homogeneous samples illusional. Third, the high variation of genotype frequencies between samples with different ethnic. Finally, the Ser9Gly polymorphism of the DRD3 gene may represent a marker rather than a causal factor.Case - control association studies detect association between an allele and a disease and can be powerful tools for gene mapping of complex disease because its samlpe is easy to collect and its result is easy to analysis.We use salting Out Procedure for Human DNA Extraction from little freezing blood. The procedure may be scaled up to handle larger samples. It is rapid, safe and inexpensive the quality of DNA is excellent. For DRD3 genotyping, PCR - RFLP showed to be a fast and and reliable diagnostic tool . It has the advantage of producing clear and distinct profiles for each speciesand useful for de-...