Study On The Polymorphisms In The Promoter Region Of Dopamine D4 Receptor Gene And Association With Chronic Tic Disorder

ObjectiveChronic tic disorder is a childhood-onset neuropsychiatric disorder characterized by multiple motor or vocal tics lasting more than one year. The prevalence of tics occurs in more males than females. In addition, affected individuals frequently display symptoms such as obsessive-compulsive disorder, attention-deficit hyperactivity disorder and behaviors disorder. Despite evidence that chronic tic disorder is an inherited disorder, the exact genetic abnormality is unknown. Dopaminergic hypotheses have included abnormalities of both pre- and postsynaptic function in chronic tic disorder. Based on neurobiological theories, most of the candidate genes belong to the dopamine neurotransmitter system. In particular, the highly polymorphic dopamine D4 receptor (DRD4) gene has attracted increasing interest.Currently, there were many studies about the 48-bp variable number of tandem repeats (VNTR) in the third exon polymorphism of the DRD4 gene and their relationship with tics, ADHD ect. neuropsychiatric disorder, but there is less reports concerning polymorphisms in the promoter region of the DRD4 gene distributing characteristics in liaoning, and association with chronic tic disorder. In our study, we test three polymorphisms in the promoter region (-1240L/S, -616C/G, -521C/T). Firstly, we study the distribution of the three gene polymorphisms in Chinese Han population Liaoning. Secondly, the association of polymorphisms in the promoter region with chronic tic disorder was studied using a case-control design. Finally, the functional activity of polymorphism was investigated through gene transfection and Dual-Luciferase Reporter assay system. To study whether there is an association of the three functional polymorphisms in the promoter region of dopamine D4 receptor (DRD4) gene with chronic tic disorder.Methods1. The three gene polymorphisms in the promoter of Dopamine D4 receptor in Chinese Han population LiaoningGenomic DNA was isolated from venous blood leukocytes from 100 healthy unrelated individuals. Polymorphisms of DRD4, -1240L/S, -616C/G and -521C/T, were genotyped by PCR and allele specific amplification (ASA) techniques and tested Hardy-Weinberg equilibrium. Current results were compared with the data on other ethnic groups, and carried X~2 test.2. Association between the polymorphisms in the promoter region of dopamine D4 receptor gene and chronic tic disorderGenomic DNA was isolated from venous blood leukocytes from 84 unrelated patients with chronic tic disorder (Study group) and 100 healthy unrelated individuals as controls. Polymorphisms of DRD4, -1240L/S, -616C/G and -521C/T, were genotyped by allele-specific primer (ASP) PCR. The frequencies of genotypes, allele and haplotypes were analysed by SHEsis online.3. The functional study of-616C/G in the promoter region of DRD4 gene Particular geneomes of two homozygotes of -616C/G, -616CC and -616GG respectively were templates in above-mentioned experiment. We constructed luciferase reporter gene express vectors of -616C/G in the promoter region of DRD4 gene, pGL3-D4Cand pGL3-D4G, and respetively. Constructed vectors pGL3-D4C and pGL3-D4G were transfected into HeLa cell through lipid vector. The transcriptional activity of-616C/G was determined by Dual-Luciferase reporter assay.Results1. The three gene polymorphisms in the promoter of Dopamine D4 receptor in Chinese Han population LiaoningWithin the 100 individuals tested, the frequencies of -1240L/S in the 5'promoter region of the DRD4 gene were found to be 60% and 40%, respectively, which met Hardy-Weinberg equilibrium. The frequencies of -616C/G were found to be 18% and 82%, respectively, which met Hardy-Weinberg equilibrium. The frequencies of -521C/T were found to be 39% and 61%, respectively, which met Hardy-Weinberg equilibrium. Chinese Liaoning populations comparing with other populations gene polymorphism distribute frequency contrasting studying find that -1240L/S no significant difference in allele frequencies between Liaoning and shanghai, but significant difference between Liaoning and Japan, Europe and Canada. There was significant difference in allele distributions of -616C/G of DRD4 between the Liaoning Han population and those of shanghai Japan, Europe and Hungary. The distribution of -521C/T of DRD4 gene has not exhibit ethnic heterogeneity.2. Association between the polymorphisms in the promoter region of dopamine D4 receptor gene and chronic tic disorderThere were no significant differences in allele frequencies and genotypes frequencies of DRD4 -1240L/S and -521C/T between the Study and the Control groups. There were significant differences in allele frequencies and genotypes frequencies (x~2 =8.419, P<0.01; x~2=7.860, P<0.05 respectively) of DRD4-616C/G between the Study and the Control groups. The haplotypic frequencies of LCT (-1240L/S, -616C/G, -521C/T) in the Study group were noticeably higher than in the Control group (x~2 =6.371, P<0.05).3. The functional study of -616C/G in the promoter region of DRD4 gene We had constructed pGL3-D4C and pGL3-D4G vector successfully. Transfection efficiencies were normalized by cotransfection with the Renilla vector, pRL-TK. Both pGL3-basic and pGL3-Control were separately transfected into the HeLa cells, which served as negative and positive controls, respectively, within the assay. The transcriptional activity of -616C/G was determined by Dual- Luciferase reporter assay. The transcriptional luciferase activety of pGL3-D4C and pGL3-D4G wre calculated by taking the ratio of the firefly luciferase activity versus the Renilla luciferase activity. Significant differences in luciferase activity were observed between pGL3-D4C and pGL3-D4G constructs at p<0.05, There were transcriptional activity of -616C and -616G. These ratios were compared with that obtained for the pGL3-basic vector to produce fold values representing increments in transcriptional activity over basic for pGL3-D4C and pGL3-D4G as follows: 7.6±0.5, 34.7±1.5. The transcriptional activity of -616C was much lower than that of -616G, The transcriptional activity of -616G was 4.6 times to -616C.Conclusion1. The distributions of -1240L/S and -616C/G of DRD4 gene exhibit ethnic heterogeneity; but .the distribution of -521C/T of DRD4 gene has not exhibit ethnic heterogeneity.2. There is an association between the DRD4-616C/G polymorphism and chronic tic disorder. The individuals with haplotype LCT (-1240L/S, -616C/G, -521C/T) are more susceptible to chronic tic disorder.3. -616C/G may have a role in regulating the expression of the DRD4 gene.