Services on Demand
Print version ISSN 0004-282XOn-line version ISSN 1678-4227
Arq. Neuro-Psiquiatr. vol.62 no.4 São Paulo Dec. 2004
Ausência de associação entre o polimorfismo VNTR do gene do transportador de dopamina (SLC6A3) e esquizofrenia em uma população brasileira
Quirino CordeiroI; Michael TalkowskiII; Joel WoodII; Eliza IkenagaI; Homero ValladaI
IDepartment of Psychiatry, University of Sao Paulo School of Medicine, São Paulo SP, Brazil
IIDepartment of Psychiatry, University of Pittsburgh School of Medicine, Pittsburgh, USA
A role of dopaminergic dysfunction has been postulated in the aetiology of schizophrenia. We hypothesized that variations in the dopamine transporter gene (SLC6A3) may be associated with schizophrenia. We conducted case-control and family based analysis on the polymorphic SLC6A3 variable number tandem repeat (VNTR) in a sample of 220 schizophrenic patients, 226 gender and ethnic matched controls, and 49 additional case-parent trios. No differences were found in allelic or genotypic distributions between cases and controls and no significant transmission distortions from heterozygous parents to schizophrenic offspring were detected. Thus, our results do not support an association of the SLC6A3 VNTR with schizophrenia in our sample.
Key words: DAT1, SLC6A3, schizophrenia, genetic polymorphism, genetic association, trios.
Genes do sistema dopaminérgico são de escolha para a pesquisa de susceptibilidade para a esquizofrenia. Desse modo, possível contribuição do polimorfismo do gene do transportador de dopamina (SLC6A3) no aumento da vulnerabilidade para a esquizofrenia foi investigada no presente estudo. Analisou-se a distribuição do sítio polimórfico do gene do transportador de dopamina (VNTR) em uma população de 220 pacientes com esquizofrenia (critério diagnóstico: DSM-IV) e comparou-se com a distribuição em uma população controle de 226 indivíduos pareados para sexo e etnia. Nenhuma diferença foi observada na distribuição dos alelos entre casos e controles. O mesmo polimorfismo também foi investigado em uma segunda amostra composta por 49 trios (pais e probando). O resultado também foi negativo. Tais dados não dão suporte para a participação do polimorfismo do gene do transportador de dopamina no aumento de susceptibilidade para esquizofrenia na amostra estudada.
Palavras-chave: DAT1, SLC6A3, esquizofrenia, polimorfismo genético, associação genética, trios.
Schizophrenia (SCZ) affects some 1% of the general population. Epidemiological studies have indicated a strong genetic component in the pathogenesis of SCZ and heritability estimates as high as 80% have been reported1. Pharmacological evidence suggests an involvement of the dopaminergic system as many antipsychotic drugs block dopamine receptors in the brain and are highly effective in treating symptoms of SCZ2. Further, amphetamines and cocaine tend to provoke or exarcebate psychotic symptoms in susceptible individuals by preventing dopamine re-uptake3. L-DOPA has also been implicated in psychotic symptoms through variable release of dopamine into the synapse4. Therefore, genes involved in the dopaminergic system are potential targets for genetic association studies with SCZ5.
Polymorphisms in dopamine receptors have been widely examined, but the results have been inconclusive6-12. Another possible candidate is the dopamine transporter gene (SLC6A3 or DAT1). The dopamine transporter (SLC6A3) plays an important role in the regulation of dopamine levels and neurotransmission by mediating the active re-uptake of synaptic dopamine back into the neurons13. Two post-mortem studies on SLC6A3 binding and SCZ showed decreased striatal SLC6A3 density in chronic SCZ14,15. A recent study using positron emission tomography found lower SLC6A3 density in sites in the basal ganglia, particularly in the middle third of putamen, in chronic SCZ patients than controls. This may suggest a decreased expression of SLC6A3 in a subset of chronic SCZ patients16.
The SLC6A3 has been cloned and mapped to human chromosome 5 (5p15.3)17. A 40-bp variable number tandem repeat polymorphism (VNTR) has been reported in the 3'-untranslated region (3'-UTR) of SLC6A3, ranging from 3 to 11 copies of the repeated sequence in a non-coding region15. Linkage studies in SCZ pedigrees from Utah18,19, Italy20, Rouen, France, the Island of La Reunion21, Germany22, and India23 have all failed to demonstrate positive linkage of this VNTR to SCZ. Most of the past association studies have also reported no significant evidence for association between this SLC6A3 polymorphism and SCZ22-30. However, Persico and Macciardi (1997) showed that the SLC6A3 genotypes in SCZ patients displayed significantly enhanced homozygote (genotypes 9/9 and 10/10) and reduced heterozygote (genotype 9/10) frequencies of the most common genotypes when contrasted with controls31. An association between the10 allele and 10/10 genotype of SLC6A3 and schizoid/avoidant personality disorder has been reported in a sample of patients with distinct diagnoses, not specifically with SCZ32.
Thus, we report here the results of case-control and family based analyses of the SLC6A3 VNTR polymorphism in an ethnically diverse SCZ Brazilian sample.
All controls, parents and schizophrenic patients provided written informed consent. The ethical approval for the study was obtained from the Ethics Committee at the Hospital das Clínicas, University of São Paulo Medical School (CAPPesq).
Case-Control Study A) Patients sample: 220 patients were recruited from inpatient and outpatient services at the Institute of Psychiatry of the Hospital das Clínicas, University of São Paulo Medical School and diagnosed according to DSM-IV33 criteria for SCZ. B) Controls sample: 226 sex and ethnic matched healthy controls were recruited from the Blood Donation Service at the Hospital das Clínicas, University of São Paulo Medical School.
Parents-Offspring Trios A separate sample of SCZ patients (n = 49) with both parents available for study comprised the case-parent trios. These families were recruited from inpatient and outpatient services at the Institute of Psychiatry of the Hospital das Clínicas, University of São Paulo Medical School and diagnosed according to DSM-IV33 criteria for SCZ.
Genomic DNA was obtained from venous blood from each subject using the phenol chloroform method. Standard polymerase chain reaction (PCR) was performed in a reaction volume of 12 Ml which included 20 ng genomic DNA, STS reaction buffer 10x concentrated (160 mM (NH4)2SO4, 500 mM Tris-HCl, pH 9.2, 0.1 mM EDTA, 20 $ DMSO, 1% Tween 20), 5 pmol of each primer, 200 mM d NTP's, 1 unit of TaqDNA polymerase (STS), 1.5 mM MgCl2. Primer sequences were: 5'TGTGGTGTAGGGA CGGCCTGAG-3' (forward) and 5'CTTCCTGGAGGTCACGGCTCAAGC-3' (reverse). Amplification consisted of 94ºC for 3 min, touchdown PCR conditions using primer annealing temperatures of 66ºC and 64ºC at two cycles each, followed by 30 cycles of 94ºC for 30 s, 62ºC for 45 s and 72ºC for 45 s and a final extension step at 72ºC for 10 min. The fragment sizes were: 280 bp (five repeats), 320 bp (six repeats), 360 bp (seven repeats), 400 bp (eight repeats), 440 bp (nine repeats), 480 bp (10 repeats), 520 bp (11 repeats), 600 bp (13 repeats). The amplification products were resolved on 1% agarose/1.5% Metaphore gels and visualized by ethidium bromide transillumination. Alleles ranging from 3 to 11 repeats were observed.
In the case-control design, the allelic and genotypic distribution in SCZ patients and health controls were contrasted using the CLUMP Program34. For family based analysis of case-parent trios, the preferential transmission of alleles from parents to affected offspring was analysed by the Extended Transmission Disequilibrium Test (ETDT), which performs the test for markers with multiple alleles35. The p values were corrected for multiple testing using Monte Carlo ETDT (MCETDT)36. Hardy- Weinberg equilibrium was calculated using the STATA Program37.
There were no significant deviations from the Hardy-Weinberg equilibrium in any of the populations for the polymorphism studied. Case-control analysis provided no evidence for allelic or genotypic association of SLC6A3 VNTR polymorphism and SCZ (Table 1). Family based analyses revealed no significant preferential transmission for any of the alleles (allele-wise TDT, c2=5.36; 4df; p=0.25) or genotypes (genotype-wise TDT, c2=5.39; 4df; p=0.24). When correcting our results for multiple testing we used MCETDT program and obtained values of p=0.43 for both allelic and genotypic transmission (Table 2).
Past findings of post-mortem and neuroimaging studies have suggested that SLC6A3 may play a role in the pathophysiology of SCZ14,15,16. However, most genetic studies investigating differences in SCZ cases and controls of this VNTR polymorphism at SLC6A3 have failed to find an association with the disorder18-30. However, one previous study has reported that the SLC6A3 genotypes in SCZ patients displayed significantly enhanced homozygote (genotypes 9/9 and 10/10) and reduced heterozygote (genotype 9/10) frequencies of the most common genotypes, maybe representing stigmata of assortative mating31.
Thus, we conducted both case-control and family based analyses of the VNTR polymorphism in a large sample of SCZ cases as well as a smaller sample of case-parent trios. We compared the frequencies of alleles and genotypes between 220 cases and 226 controls and did not find significant differences. Our family based analysis also failed to detect linkage or association of this polymorphism with SCZ.
Distinct racial and ethnic groups display significant differences in SLC6A3 marker distributions38. Thus, case-control association studies can potentially be underpowered to detect association in a sample of this size, particularly in the presence of ethnic admixture. This potential confound may be critical in populations of high ethnic admixture such as a Brazilian population8,39. Therefore, we subsequently performed family based analyses of transmission distortions from heterozygous parents to SCZ offspring. This type of analysis avoids stratification biases40. Thus, our family based analysis may provide more conclusive evidence of a lack of association at this locus with SCZ.
This finding is in accordance with most of the studies conducted to date on this polymorphism in the dopamine transporter gene. However, the SLC6A3 VNTR polymorphism may be involved in the susceptibility for other psychotic disorders, such as bipolar disorder41-43. Further, cocaine acts on SLC6A3, enhancing the dopaminergic transmission and making this gene a strong candidate in cocaine-induced paranoia44. Future analysis on larger populations are also required to determine if other variations in the dopamine transporter provide evidence for association to SCZ.
We would like to thank Prof. Vishwajit Nimgaonkar at the University of Pittsburgh.
1. McGuffin P, Owen MJ, O'Donovan MC, Thapar A, Gottesman I. Seminars in psychiatric genetics. London: Royal College of Psychaitrists, 1994. [ Links ]
2. Frederickson A. The dopamine hypothesis of schizophrenia. Second Web Rep 1998. [ Links ]
3. Goodwin FK, Jamison KR. Manic-depressive illness. New York: Oxford University Press, 1990. [ Links ]
4. Murphy DL, Brodie HK, Goodwin FK, Bunney WE. Regular induction of hypomania by L-DOPA in bipolar manic-depressive patients. Nature 1971;229:135-136. [ Links ]
5. Brown AS, Gershon S. Dopamine and depression. J Neural Transm Genet Sec 1993;91:75-109. [ Links ]
6. Emilien G, Maloteaux JM, Geurts M, Owen MJ. Dopamine receptors and schizophrenia: contribution of molecular genetics and clinical neuropsychology. Int J Neuropsychopharm 1999;2:197-227. [ Links ]
7. Cordeiro Q Jr, Junqueira R, Vallada H. Study of association between the ser-9-gly polymorphism of the D3 dopaminergic receptor and schizophrenia. Arq Neuropsiquiatr 2001;59:219-222. [ Links ]
8. Lung FW, Tzeng DS, Shu BS. Ethnic heterogeneity in allele variation in the DRD4 gene in schizophrenia. Schizophr Res 2002;57:239-245. [ Links ]
9. Glatt SJ, Faraone SV, Tsuang MT. Meta-analysis identifies an association between the dopamine D2 receptor gene and schizophrenia. Mol Psychiatry 2003;8:911-915. [ Links ]
10. Jonsson EG, Sillen A, Vares M, Ekholm B, Terenius L, Sedvall GC. Dopamine D2 receptor gene Ser311Cys variant and schizophrenia: association study and meta-analysis. Am J Med Genet 2003;119:28-34. [ Links ]
11. Jonsson EG, Flyckt L, Burgert E, et al. Dopamine D3 receptor gene Ser9Gly variant and schizophrenia: association study and meta-analysis. Psychiatr Genet 2003;13:1-12. [ Links ]
12. Jonsson EG, Sedvall GC, Nothen MM, Cichon S. Dopamine D4 receptor gene (DRD4) variants and schizophrenia: meta-analyses. Schizophr Res 2003;61:111-119. [ Links ]
13. Jaber M, Jones S, Giros B, Caron MG. The dopamine transporter: a crucial component regulating dopamine transmission. Movement Disord 1997;12:629-633. [ Links ]
14. Laakso A, Bergman J, Haaparanta M, et al. Decreased striatal dopamine transporter binding in vivo in chronic schizophrenia. Schizophr Res 2001;52:115-120. [ Links ]
15. Knable MB, Hyde TM, Herman MM, Carter JM, Bigelow L, Kleinman JE. Quantitative autoradiography of dopamine-D1 receptors, D2 receptors, and dopamine uptake sites in postmortem striatal specimens from schizophrenic patients. Biol Psychiatry 1994;36:827-835. [ Links ]
16. Chinaglia G, Alvarez FJ, Probst A, Palacios JM. Mesostriatal and mesolimbic dopamine uptake binding sites are reduced in Parkinson's disease and progressive supranuclear palsy: a quantitative autoradiographic study using [3H]mazindol. Neuroscience 1992;49:317-327. [ Links ]
17. Giros B, Mestikawy S, Godinot N, et al. Cloning, pharmacological characterization, and chromosome assignment of the human dopamine transporter. Mol Pharmacol 1992;42:383-390. [ Links ]
18. Byerley W, Coon H, Hoff M, et al. Human dopamine transporter gene not linked with schizophrenia in multigenerational pedigrees. Hum Hered 1993;43:319-322. [ Links ]
19. Byerley W, Hoff M, Holik J, Caron MG, Giros B. VNTR polymorphism for the human dopamine transporter gene (DAT1). Hum Mol Genet 1993;2:335. [ Links ]
20. Persico AM, Wang ZW, Black DW, Andreasen NC, Uhl GR, Crowe RR. Exclusion of close linkage of the dopamine transporter gene with schizophrenia spectrum disorders. Am J Psychiatry 1995;152:134-136. [ Links ]
21. Bodeau-Pean S, Laurent C, Campion D, et al. No evidence for linkage or association between the dopamine transporter gene and schizophrenia in a French population. Psychiatry Res 1995;59:1-6. [ Links ]
22. Maier W, Minges J, Eckstein N, et al. Genetic relationship between dopamine transporter gene and schizophrenia: linkage and association. Schizophr Res 1996;20:175-180. [ Links ]
23. Semwal P, Prasad S, Bhatia T, et al. Family-based association studies of monoaminergic gene polymorphisms among North Indians with schizophrenia. Mol Psychiatry. 2001;6:220-224. [ Links ]
24. Li T, Yang L, Wiese C. No association between alleles or genotypes at the dopamine transporter gene and schizophrenia. Psychiatry Res 1994;52:17-23. [ Links ]
25. Daniels J, Williams J, Asherson P, McGuffin P, Owen M. No association between schizophrenia and polymorphisms within the genes for debrisoquine 4-hydroxylase (CYP2D6) and the dopamine transporter (DAT). Am J Med Genet 1995;60:85-87. [ Links ]
26. Joober R, Toulouse A, Benkelfat C, et al. DRD3 and DAT1 genes in schizophrenia: an association study. J Psychiatr Res 2000;34:285-291. [ Links ]
27. Inada T, Sugita T, Dobashi I, et al. Dopamine transporter gene polymorphism and psychiatric symptoms seen in schizophrenic patients at their first episode. Am J Med Genet 1996;26:406-408. [ Links ]
28. Georgieva L, Dimitrova A, Nikolov I, et al. Dopamine transporter gene (DAT1) VNTR polymorphism in major psychiatric disorders: family-based association study in the Bulgarian population. Acta Psychiatr Scand 2002;105:396-399. [ Links ]
29. Hauser J, Kapelski P, Czerski PM, et al. Lack of association between VNTR polymorphism of DAT gene and schizophrenia. Psychiatr Pol 2002;36:403-412. [ Links ]
30. Semwal P, Prasad S, Varma PG, Bhagwat AM, Deshpande SN, Thelma BK. Candidate gene polymorphisms among North Indians and their association with schizophrenia in a case-control study. J Genet 2002;81:65-71. [ Links ]
31. Persico AM, Macciardi F. Genotypic association between dopamine transporter gene polymorphisms and schizophrenia. Am J Med Genet 1997;74:53-57. [ Links ]
32. Blum K, Braverman ER, Wu S, et al. Association of polymorphisms of dopamine D2 receptor (DRD2), and dopamine transporter (DAT1) genes with schizoid/avoidant behaviors (SAB). Mol Psychiatry 1997;2:239-246. [ Links ]
33. American Psychiatric Association. Diagnostic and statistical manual of mental disorders, 4th edn. Washington, DC: American Psychiatric Association, 1994. [ Links ]
34. Sham PC, Curtis D. Monte Carlo tests for associations between disease and alleles at highly polymorphic loci. Ann Hum Genet 1995;59:97-105. [ Links ]
35. Sham PC, Curtis D. An extended transmission / disequilibrium test (TST) for multi-allele marker loci. Ann Hum Genet 1995;59:323-336. [ Links ]
36. North BV, Curtis D, Sham PC. A note on the calculation of empirical p-values from Monte Carlo procedures. Am J Hum Genet 2002;71:445-7. [ Links ]
37. Shan P. Statistics in human genetics. New York: Arnold, 1998. [ Links ]
38. Persico AM, Bird G, Gabbay FH, Uhl GR. D2 dopamine receptor gene TaqI A1 and B1 restriction fragment length polymorphisms: enhanced frequencies in psychostimulant-preferring polysubstance abusers. Biol Psychiatry 1996;40:776-784. [ Links ]
39. Parra FC, Amado RC, Lambertucci JR, Rocha J, Antunes CM, Pena SD. Color and genomic ancestry in Brazilians. Proc Natl Acad Sci U S A 2003;100:177-182. [ Links ]
40. Spielman R, Ewens W. The T.D.T. and other family-based tests for linkage disequilibrium and association. Am J Hum Genet 1996;59:983-989. [ Links ]
41. Kelsoe JR, Sadovnick AD, Krisbjarnarson H. Possible locus for bipolar disorder near the dopamine transporter on chromosome 5. Am J Med Genet 1996;67:533-540. [ Links ]
42. Waldman ID, Robinson BF, Feigon SA. Linkage disequilibrium between the dopamine transporter gene (DAT1) and bipolar disorder: extending the transmission disequilibrium test (TDT) to examine genetic heterogeneity. Genet Epidemiol 1997;14:699-704. [ Links ]
43. Greenwood TA, Alexander M, Keck PE, et al. Evidence for linkage disequilibrium between the dopamine transporter and bipolar disorder. Am J Med Genet 2001;105:145-151. [ Links ]
44. Gelernter J, Kranzler HR, Satel SL, Rao PA. Genetic association between dopamine transporter protein alleles and cocaine-induced paranoia. Neuropsychopharmacology 1994;11:195-199. [ Links ]
Dr. Homero Vallada
Instituto de Psiquiatria do Hospital das Clínicas/FMUSP
Sao Paulo SP - Brasil
Received 26 January 2004, received in final form 7 May 2004. Accepted 8 July 2004.