tourette syndrome ( ts ) , also known as multiple transient tic disorder or combined vocal and multiple motor tics , is a type of chronic neuropsychiatric developmental disorder that occurs in children and adolescents . it is characterized by involuntary vocal tics and multiple motor tics of the head and body , particularly the face and extremities . these symptoms may occur simultaneously or during different disease periods , showing a sudden and rapid onset , purposelessness , stereotypy , and arhythmicity . the prevalence of ts has increased within recent years and is estimated to be 0.05%3% ; a large - scale epidemiological survey of 420,312 children and adolescents reported it to be 1.0% . the ratio of the ts prevalence rate between males and females is 35:1 , and most patients develop the disease between the ages of 2 and 15 years , with a mean age of 7 years . tic severity is closely related to age , with the most severe symptoms usually occurring at 812 years of age , causing a great impact on the children 's self - esteem , self - confidence , peer relationships , and family relationships . about half of ts patients suffer social dysfunctions such as learning difficulties and interpersonal difficulties . it is widely acknowledged that ts results from the interaction between genetic and environmental factors , as shown by pedigree and twin studies . indeed , antipsychotic agents , both typical dopamine antagonists and atypical dopamine antagonists , have been proven to improve the symptoms of ts . in addition , neuroimaging studies have found abnormalities in brain structures that are functionally related to the dopamine neurotransmission system . therapeutic response to dopamine antagonists , as well as neuroimaging studies , supports a dopaminergic system abnormality in ts . currently , the polymorphism of many dopaminergic system - related genes has been identified , which may be associated with ts . the genes are the dopamine transporter 1 ( dat1 ) gene , the three d2-like dopamine receptor ( drd2 , drd3 , and drd4 ) genes and the monoamine oxidase - a gene . still , a lack of replicated and consistent results was observed , a single nucleotide polymorphisms ( snps ) in these genes have a poor reproducibility . moreover , existing studies have focused on drd2 , drd4 , and dat1 , while little is known about drd3 . in the past 10 years , we had focused on the study of exploring the association between the gene drd2 , drd4 , and dat between ts in a han chinese population . there has not been any report about the association study of ts and drd3 gene in han chinese population . in this study , we investigated the presence of the drd3 rs6280 polymorphism in ts patients , their unaffected parents , and normal controls , compared genotype and allele frequencies between ts patients and controls , and observed transmission disequilibrium between ts patients and their unaffected parents . a total of 160 ts patients was recruited from the department of pediatrics , beijing anding hospital , capital medical university , china between december 2009 and december 2012 , including 101 nuclear pedigrees . psychiatric diagnoses were made by a psychiatrist according to the diagnostic criteria of the diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) . the following inclusion criteria were used for the ts group : ( 1 ) meeting the diagnostic criteria of dsm - iv for ts ; ( 2 ) an age of 618 years ; ( 3 ) having no other central nervous system diseases or serious physical illness ; ( 4 ) a chinese han ; ( 5 ) having no tic disorder or other mental disorder patients in three generations of two pedigrees ; ( 6 ) being able to complete all diagnostic tests ; ( 7 ) providing informed consent . the following inclusion criteria were used for the control group : ( 1 ) a healthy child or adolescent ; ( 2 ) an age of 618 years ; ( 3 ) having no central nervous system disease or severe physical illness , and no drug abuse or dependence ; ( 4 ) having no mental disorder , including attempted suicide , or family history of mental disorder ; ( 5 ) a chinese han individual unrelated to other controls or ts patients ; ( 6 ) a similar gender ratio to ts patients ; ( 7 ) being able to complete all diagnostic tests ; ( 8) providing informed consent . the following inclusion criteria were used for the parents of ts patients : ( 1 ) biological parents of the ts patients ; ( 2 ) healthy individuals with no history of mental disorder and not meeting the dsm - iv diagnostic criteria ; ( 3 ) providing informed consent . this experiment was approved by the ethics committee , beijing anding hospital , capital medical university , china . the study was explained to all subjects prior to experimentation , and informed consent was obtained from all patients and their families . the consent forms were signed by the guardians of subjects < 7 years old , and by both guardians and subjects 7 years old . the work in this paper has been carried out in accordance with the code of ethics of the world medical association ( declaration of helsinki ) . fasting peripheral blood samples ( 5 ml ) were collected in edta tubes , and genomic dna was extracted using the centrifugation column method with the whole blood genomic dna extraction kit ( promega ; madison , wi , usa ) according to the manufacturer 's instructions . based on chinese han population genetics data from the database of snps ( http://www.ncbi.nlm.nih.gov/snp/ ; the us national center for biotechnology information ) , the international hapmap project ( http://www.hapmap.org/ ) , and previous studies , the drd3 snp rs6280 locus was selected for gene polymorphism analysis . dopamine d3 receptor snp rs6280 genotyping was carried out using a taqman snp genotyping probe . polymerase chain reaction ( pcr ) reactions ( 5 l ) contained 10 ng dna template , 2.5 l  2 taqman universal pcr master mix ( abi ) , and 0.25 l  20 snp assay ( applied biosystems , bedford , ma , usa ; including primers and fam / vic probe ) . the reaction conditions were 95c for 10 min followed by 40 cycles of 92c for 15 s and 60c for 1 min . pcr signals and fluorescence signals were read on an abi7900ht sequence detector ( 384-well plates ) , and then analyzed using the sds2.4 image analysis software ( abi prism , abi corp ) allelic discrimination program . fam and vic fluorescence intensities of different alleles were analyzed to determine whether samples were homozygous wild - type , heterozygous or homozygous mutated . spss 17.0 software ( ibm spss statistics for windows , ibm corp . , armonk , ny , usa ) was used for data analysis . the fitness of the genotype and allele was tested using the hardy  weinberg equilibrium ( hwe ) test , and polymorphism differences between the ts patient and control groups were analyzed using the chi - squared (  ) test . the transmission disequilibrium test ( tdt ) of nuclear pedigrees was performed using plink1.07 software ( shaun purcell , usa ) ,  = 0.05 . a total of 160 ts patients was recruited from the department of pediatrics , beijing anding hospital , capital medical university , china between december 2009 and december 2012 , including 101 nuclear pedigrees . psychiatric diagnoses were made by a psychiatrist according to the diagnostic criteria of the diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) . the following inclusion criteria were used for the ts group : ( 1 ) meeting the diagnostic criteria of dsm - iv for ts ; ( 2 ) an age of 618 years ; ( 3 ) having no other central nervous system diseases or serious physical illness ; ( 4 ) a chinese han ; ( 5 ) having no tic disorder or other mental disorder patients in three generations of two pedigrees ; ( 6 ) being able to complete all diagnostic tests ; ( 7 ) providing informed consent . the following inclusion criteria were used for the control group : ( 1 ) a healthy child or adolescent ; ( 2 ) an age of 618 years ; ( 3 ) having no central nervous system disease or severe physical illness , and no drug abuse or dependence ; ( 4 ) having no mental disorder , including attempted suicide , or family history of mental disorder ; ( 5 ) a chinese han individual unrelated to other controls or ts patients ; ( 6 ) a similar gender ratio to ts patients ; ( 7 ) being able to complete all diagnostic tests ; ( 8) providing informed consent . the following inclusion criteria were used for the parents of ts patients : ( 1 ) biological parents of the ts patients ; ( 2 ) healthy individuals with no history of mental disorder and not meeting the dsm - iv diagnostic criteria ; ( 3 ) providing informed consent . this experiment was approved by the ethics committee , beijing anding hospital , capital medical university , china . the study was explained to all subjects prior to experimentation , and informed consent was obtained from all patients and their families . the consent forms were signed by the guardians of subjects < 7 years old , and by both guardians and subjects 7 years old . the work in this paper has been carried out in accordance with the code of ethics of the world medical association ( declaration of helsinki ) . fasting peripheral blood samples ( 5 ml ) were collected in edta tubes , and genomic dna was extracted using the centrifugation column method with the whole blood genomic dna extraction kit ( promega ; madison , wi , usa ) according to the manufacturer 's instructions . based on chinese han population genetics data from the database of snps ( http://www.ncbi.nlm.nih.gov/snp/ ; the us national center for biotechnology information ) , the international hapmap project ( http://www.hapmap.org/ ) , and previous studies , the drd3 snp rs6280 locus was selected for gene polymorphism analysis . dopamine d3 receptor snp rs6280 genotyping was carried out using a taqman snp genotyping probe . polymerase chain reaction ( pcr ) reactions ( 5 l ) contained 10 ng dna template , 2.5 l  2 taqman universal pcr master mix ( abi ) , and 0.25 l  20 snp assay ( applied biosystems , bedford , ma , usa ; including primers and fam / vic probe ) . the reaction conditions were 95c for 10 min followed by 40 cycles of 92c for 15 s and 60c for 1 min . pcr signals and fluorescence signals were read on an abi7900ht sequence detector ( 384-well plates ) , and then analyzed using the sds2.4 image analysis software ( abi prism , abi corp ) allelic discrimination program . fam and vic fluorescence intensities of different alleles were analyzed to determine whether samples were homozygous wild - type , heterozygous or homozygous mutated . spss 17.0 software ( ibm spss statistics for windows , ibm corp . , armonk , ny , usa ) was used for data analysis . the fitness of the genotype and allele was tested using the hardy  weinberg equilibrium ( hwe ) test , and polymorphism differences between the ts patient and control groups were analyzed using the chi - squared (  ) test . the transmission disequilibrium test ( tdt ) of nuclear pedigrees was performed using plink1.07 software ( shaun purcell , usa ) ,  = 0.05 . comparison of general information a total of 160 sporadic ts patients ( including 101 ts patients ) involved in pedigree studies and 59 unrelated cases underwent drd3 snp rs6280 analysis . of the 90 normal controls , 73 were male and 17 were female , at a ratio of 4.3:1 . there was no significant difference in the gender ratio ( p = 0.164 ) or mean age ( p = 0.927 ) between the two groups . fitness of hardy  weinberg equilibrium test dopamine d3 receptor rs6280 genotype frequencies achieved hwe in the control group ( p = 0.074 ) and ts patient group ( p = 0.582 ) , as shown by the -test . this indicated that the data are reliable and representative for genetic association analysis [ table 1 ] . drd3 rs6280 hardy - weinberg equilibrium test in the control and ts patient groups drd3 : dopamine d3 receptor ; ts : tourette syndrome . comparison of dopamine d3 receptor 3 gene rs6280 genotype and allele frequencies the frequencies of drd3 rs6280 genotypes c / c , c / t , and t / t were 10.6 , 46.9 , and 42.5% , respectively , in the ts patient group , and 13.3% , 34.4% , and 52.2% , respectively , in the control group . the frequencies of c and t alleles were 34.1% and 65.9% , respectively , in the ts patient group , and 30.6% and 69.4% , respectively , in the control group . there were no significant differences in genotype or allele frequencies between the ts patient and control groups ( p = 0.161 and 0.423 , respectively ; table 2 ) . comparison of genotype and allele frequencies between the ts patient and control groups ts : tourette syndrome . comparison of general information the nuclear pedigree study included ts patients ( n = 101 ) and their biological parents ( n = 202 ) . of the ts patients , 80 were male , and 21 were female , at a ratio of 3.8:1 . fitness of hardy  weinberg equilibrium test dopamine d3 receptor rs6280 genotype frequencies achieved hwe in both the ts patient group ( p = 0.805 ) and parents group ( p = 0.727 ) , as shown by the -test [ table 3 ] . drd3 rs6280 hardy - weinberg equilibrium test in the ts patient and parents groups ts : tourette syndrome . comparison of genotype and allele frequencies in the tourette syndrome patient and parents groups the frequencies of genotypes c / c , c / t , and t / t were 10.9% , 42.6% , and 46.5% , respectively , in the ts patient group , and 23.8% , 43.6% , and 32.7% , respectively , in the parents group . the c allele frequency was 32.2% and the t allele frequency 67.8% in both groups [ table 4 ] . comparison of genotype and allele frequencies in the ts patient and parents groups the number in brackets refers to the frequency ( % ) . transmission disequilibrium test analysis revealed no transmission disequilibrium in the drd3 rs6280 snp among ts nuclear pedigrees , indicating that this locus is not associated with ts ( p = 1 ; table 5 ) . tdt analysis in ts pedigree or : odds ratio ; ci : confidence interval ; ts : tourette syndrome ; tdt : transmission disequilibrium test . comparison of general information a total of 160 sporadic ts patients ( including 101 ts patients ) involved in pedigree studies and 59 unrelated cases underwent drd3 snp rs6280 analysis . of the 90 normal controls , 73 were male and 17 were female , at a ratio of 4.3:1 . there was no significant difference in the gender ratio ( p = 0.164 ) or mean age ( p = 0.927 ) between the two groups . fitness of hardy  weinberg equilibrium test dopamine d3 receptor rs6280 genotype frequencies achieved hwe in the control group ( p = 0.074 ) and ts patient group ( p = 0.582 ) , as shown by the -test . this indicated that the data are reliable and representative for genetic association analysis [ table 1 ] . drd3 rs6280 hardy - weinberg equilibrium test in the control and ts patient groups drd3 : dopamine d3 receptor ; ts : tourette syndrome . comparison of dopamine d3 receptor 3 gene rs6280 genotype and allele frequencies the frequencies of drd3 rs6280 genotypes c / c , c / t , and t / t were 10.6 , 46.9 , and 42.5% , respectively , in the ts patient group , and 13.3% , 34.4% , and 52.2% , respectively , in the control group . the frequencies of c and t alleles were 34.1% and 65.9% , respectively , in the ts patient group , and 30.6% and 69.4% , respectively , in the control group . there were no significant differences in genotype or allele frequencies between the ts patient and control groups ( p = 0.161 and 0.423 , respectively ; table 2 ) . comparison of genotype and allele frequencies between the ts patient and control groups ts : tourette syndrome . comparison of general information the nuclear pedigree study included ts patients ( n = 101 ) and their biological parents ( n = 202 ) . of the ts patients , fitness of hardy  weinberg equilibrium test dopamine d3 receptor rs6280 genotype frequencies achieved hwe in both the ts patient group ( p = 0.805 ) and parents group ( p = 0.727 ) , as shown by the -test [ table 3 ] . drd3 rs6280 hardy - weinberg equilibrium test in the ts patient and parents groups ts : tourette syndrome . comparison of genotype and allele frequencies in the tourette syndrome patient and parents groups the frequencies of genotypes c / c , c / t , and t / t were 10.9% , 42.6% , and 46.5% , respectively , in the ts patient group , and 23.8% , 43.6% , and 32.7% , respectively , in the parents group . the c allele frequency was 32.2% and the t allele frequency 67.8% in both groups [ table 4 ] . comparison of genotype and allele frequencies in the ts patient and parents groups the number in brackets refers to the frequency ( % ) . transmission disequilibrium test analysis revealed no transmission disequilibrium in the drd3 rs6280 snp among ts nuclear pedigrees , indicating that this locus is not associated with ts ( p = 1 ; table 5 ) . tdt analysis in ts pedigree or : odds ratio ; ci : confidence interval ; ts : tourette syndrome ; tdt : transmission disequilibrium test . current molecular genetics methods for the detection of susceptible genes in complex diseases include linkage analyses and association studies , the latter comprising either case  control or pedigree studies . case control studies compare differences in genotype and allele frequencies at candidate loci between cases and controls , so analyze the data of all subjects . however , they are limited in that the association analysis is affected by population genetic stratification and other factors . therefore , many association studies are difficult to repeat in different population . for this reason , pedigree studies are often preferred . pedigree studies effectively avoid the interference of population stratification ; however , the efficiency of tdt studies has been shown to be lower than the case  control studies . the two types of analyses can complement each other , enabling the same genetic marker to be detected by both methods with more reliable results . dopamine d3 receptor is mainly located in the region of the brain associated with cognition and affection , and the drd3 receptor is highly expressed in the mesolimbic dopamine system including the extended amygdala . the drd3 ser9gly polymorphism ( rs6280 ) involves a serine to glycine substitution that may affect the d3 receptor affinity . it has been widely used in studies addressing the genetic causes of neuropsychiatric disorders , including an investigation of susceptible loci correlations with psychiatric disease . comings et al . previously identified an association between ts and homozygosity at the drd3 gene , but this was not replicated in two studies published in the same year . demonstrated linkage disequilibrium in drd3 polymorphism loci bali and mspi , which was not associated with ts in 16 pedigrees . found no association between ts and drd3 in 110 nuclear pedigrees by tdt analysis , while kowalska et al . there has not been any report about the association study of ts and drd3 gene polymorphisms in han chinese population . this study aimed to explore the association between the drd3 gene rs6280 and ts using both case  controlled study and nuclear pedigrees tdt analysis . the normal controls were well - matched with ts patients with respect to race , region , gender , and age , thus minimizing the influence of race , geography , and population stratification on experimental results , and increasing the reliability of our findings . we found no significant differences in genotype or allele frequencies between healthy controls and ts patients , while tdt analysis also revealed no transmission disequilibrium in nuclear pedigrees . few efforts have directly focused on the correlation between ts and drd3 , and this study was the first to do so in a chinese han population . our combination of a case  control study and nuclear pedigrees tdt analysis produced a more reliable result , which suggests that the drd3 snp rs6280 may be not associated with ts in a han chinese population . the main is inconsistent with research method and different study populations . at the same time , ts is a heterogeneous disorder , often comorbidity with attention deficit hyperactivity disorder and obsessive - compulsive disorder . our findings provide no evidence for an association between drd3 gene rs6280 and ts in the han chinese population . it should be verified by making larger samples and making further efforts to subtype ts in future study .