Zhaoyun Du, Guangxin Wang, Yuebing Zhang, Yiren Cheng, Chuan’an Zhu

1Department of Laboratory Medicine, Jining Medical College, Jining 272067, Shandong Province, China

2Institute of Pediatrics, Qilu Children’s Hospital, Shandong University, Jinan 250022, Shandong Province, China

3Department of Psychiatry, Second Affiliated Hospital of Jining Medical College, Jining 272051, Shandong Province, China

4Department of Laboratory, First People’s Hospital of Jining, Jining 272001, Shandong Province, China

lNTRODUCTlON

Schizophrenia (MIM ID: 181500) is a common disorder with a lifetime prevalence of approximately 1%[1]. It often develops in young adults who initially seem without symptoms. Upon progression, schizophrenia is characterized by a constellation of symptoms, including hallucinations and delusions, and symptoms such as severely inappropriate emotional responses, disordered thinking and concentration, erratic behavior, as well as social and occupational deterioration. There may also be mild impairment of cognitive function[2]. It is heritable, but the genetics are complex[3-5].

Because dysfunctions in the brain dopaminergic system are involved in a variety of important disorders including schizophrenia,dopaminergic receptors are often targeted in the treatment of these disorders[6]. Dopamine D1 receptor (DRD1) is one of the dopaminergic receptors. Its encoding gene is mapped to the chromosome region 5q35.1, including 2 exons and 1 intron[7].

Several polymorphisms of this gene were identified. Besides the restriction fragment length polymorphisms recognized by EcoRI and TaqI[7], four other polymorphisms were reported, -94 G/A substitution (rs5326) and-48 A/G substitution in the 5’ untranslated region of exon 2, and 198 G/A and 1 263 G/A substitutions. Data indicating the role of these mutations in the biosynthesis of proteins are emerging. Some researchers have suggested an association between the DRD1 gene polymorphism and performance on a prefrontal cortex test for schizophrenia[8-9]. The DRD1 association showed a strong degree of epidemiologic credibility[10].

However, the question of whether the -94 G/A polymorphism of the DRD1 gene is associated with schizophrenia remains controversial[11].

We hypothesize that the -94 G/A polymorphism of the DRD1 gene may be associated with the pathogenesis of schizophrenia, as prefrontal cortex impairment is one of the most conspicuous pathological findings in this illness[12]. To test this, we genotyped this functional polymorphism in a Han population of 162 schizophrenia patients and compared it with that of 101 healthy controls in Shandong province, China.

RESULTS

Quantitative analysis of the subjects

A total of 162 patients with schizophrenia(serving as the patient group) were inpatients treated at the Department of Psychiatry, Second Affiliated Hospital of Jining Medical College, China,from January 2009 to September 2009. Meanwhile, 101 healthy controls (serving as the normal control group)were randomly selected from a group of general medical examination participants in the First People’s Hospital of Jining, China. All 162 patients with schizophrenia and 101 normal controls were included in the final analysis.

Comparison of baseline information between 162 schizophrenia patients and 101 normal controls

The baseline information of 263 subjects is described in Table 1. There were no significant differences in gender or age between patients with schizophrenia and normal controls.

Table 1 Comparison of baseline information between 162 schizophrenia patients and 101 healthy controls

-94 G/A site polymorphism analysis

The DNA samples from patients and controls were genotyped using the real-time StatMinerTMsoftware and the TaqMan SNP Genotyping Assay System. The DRD1 gene at the -94 G/A site included the G allele and the A allele, in three genotypes of G/G, G/A, and A/A. The probe of allele 1 (G), which matched with the wild type,was labeled with FAM (color red); the probe of allele 2(A), which matched with the mutant was labeled with VIC(color blue). Both are heterozygous, shown as color green (Figure 1).

Figure 1 Single nucleotide polymorphism scatter-plot of dopamine D1 receptor gene at the -94 G/A site was detected by real-time StatMiner™ software. Allele X is allele G, shown as a red dots; allele Y is allele A, shown as blue dots. Both are heterozygous, shown as green dots.

Hardy-Weinberg equilibrium at the -94 G/A site

The chi-square test was used to assess whether the SNP genotypes are in Hardy-Weinberg equilibrium between the schizophrenic patients and controls. Genotype distributions were in agreement with the Hardy-Weinberg equilibrium, and there was no significant difference between the observed value and the expected value at the-94 G/A site in both the patient group (χ2= 2.716, P =0.257) and control group (χ2= 1.921, P = 0.383; Table 2).

These experimental findings revealed that the enrolled subjects are a large population and their data are reliable.

Table 2 Hardy-Weinberg equilibrium for the patient group and control group

Genotypes and allele frequencies of the -94 G/A polymorphism in the DRD1 gene

The -94 G/A polymorphism within the DRD1 gene were genotyped in 162 patients with schizophrenia and 101 controls. The distribution of the G/A genotype was significantly different between the schizophrenia group and the control group [χ2= 4.238, P ＜ 0.05; Odds ratio (OR) =1.535, 95% CI: 0.900-2.618], but the G and A allele frequencies were not significantly different between the two groups (χ2= 0.132, P = 0.717; OR = 0.929, 95% CI:0.623-1.385; Table 3).

Table 3 Genotypes and allele frequencies of -94 G/A polymorphism in the dopamine D1 receptor gene between schizophrenic patients and healthy controls [n(%)]

These results demonstrated that the G/A genotype of the-94 G/A polymorphism within the DRD1 gene may increase the incidence of schizophrenia.

DlSCUSSlON

Schizophrenia is a severe psychiatric disorder. Many aspects of its etiology and pathophysiology remain poorly understood despite extensive research. However,the considerable contribution made by genetic factors to schizophrenia susceptibility is well recognized[13]. The heritability of schizophrenia is around 80%[14], and a first degree relative has a 5- to 10-fold increase in the risk of developing the disorder compared with the risk for the general population[15].

Neuropsychological and functional brain imaging data suggest that disordered behavior in schizophrenia is associated with aberrant function in the prefrontal cortex[16]. Dopamine, an important neurotransmitter in the prefrontal cortex, is believed to be involved in the pathogenesis of schizophrenia[17-18]. The diverse physiologic actions of dopamine are mediated by its interaction with two types of G protein-coupled receptors, DRD1 and DRD2, which stimulate and inhibit, respectively, the enzyme adenylyl cyclase.

The DRD1 gene encodes a protein of 446 amino acids,having a predicted relative molecular mass of 49 300 Da and a transmembrane topology similar to that of other G protein-coupled receptors. Controversy about whether the DRD1 gene being a schizophrenia susceptibility gene still exists despite ample evidence suggesting that the DRD1 gene is associated with performance on neuropsychological tests probing the function of the prefrontal cortex in schizophrenia, as well as its association with positive and negative symptoms. Allele et al[19]found that a DRD1 polymorphism, -48 A/G allele (rs4532), was associated with susceptibility to schizophrenia. According to the Venice guidelines for the assessment of cumulative evidence in genetic association studies, the DRD1 association showed a strong degree of epidemiologic credibility[8]. However, Zhang et al[11]genotyped five single nucleotide polymorphisms within the DRD1 gene involving 373 schizophrenia patients at early age of onset and 379 healthy subjects. No significant differences of genotype, allele, or haplotype distribution were identified between the patients and controls.

The -94 G/A polymorphism (rs5326) in the DRD1 gene exon 2, due to a G to A substitution, may act as a short open reading frame preceding the receptor cistron and encode a small peptide that inhibits receptor translation[20]. Zhang et al[11]reported no association between the DRD1 -94 G/A polymorphism and schizophrenia in a Chinese population. To further determine whether the DRD1 -94 G/A polymorphism is involved in the etiology of schizophrenia, we undertook a case-control study to look for an association. We genotyped this SNP within the DRD1; involving 162 schizophrenia patients and 101 healthy controls. Results showed that the distribution of the G/A genotype of the -94 G/A polymorphism within the DRD1 gene differed significantly in schizophrenics compared with normal controls, indicating that the G/A genotype of the -94 G/A polymorphism within the DRD1 gene may increase the incidence of schizophrenia. The inconsistency of association studies between DRD1 and schizophrenia may be due to genetic heterogeneity; a significant challenge in the genetic study of schizophrenia. The results obtained in our study may provide the evidence for a possible involvement of the DRD1 -94 G/A polymorphism in the pathogenesis of schizophrenia.

Further functional analyses and genetic studies are warranted to determine the interaction of the SNPs among the DRD1 gene, or between the DRD1 gene and other genes, as well as their possible roles in the etiology of schizophrenia.

In conclusion, the -94 G/A polymorphism in the DRD1 gene is associated with schizophrenia in a Chinese Han population living in Shan province, and the polymorphisms revealed by this study warrant further investigation.

SUBJECTS AND METHODS

Design

A case-control, genetic association study.

Time and setting

The experiment was performed at the Central Laboratory, Jining Medical College, China, from August 2009 to November 2010.

Subjects

Patient group

A total of 162 schizophrenic patients were enrolled. A consensus diagnosis, by at least two psychiatrists, was made for each patient, according to DSM-IV criteria[21].

Control group

In total, 101 healthy controls met the following criteria:(1) no symptoms or history of schizophrenia or related disorders; (2) absence of first-degree relatives with a history of schizophrenia.

All patients and healthy controls were unrelated, of Chinese Han population in Shandong province, China. They received a detailed description of the study and gave written informed consent.

Methods

Genomic DNA extraction

Venous blood, 2 mL, was collected, and genomic DNA was extracted using Puregene DNA Extraction Kit(Qiagen, Hilden, Germany). Genomic DNA was dissolved in Tris-borate elution buffer, and the concentration(mg/mL) and purity (A260nm/280nm) were measured with an ultraviolet spectrophotometer (Syngene, Frederick, MD,USA). Genomic DNA was stored at –20°C, and used as a template for PCR.

Genotypes and allele frequencies of -94 G/A polymorphism in the DRD1 gene detected by PCR

Genotyping of the DRD1 gene -94 G/A (rs5326) was performed in 384-well plates using a pre-designed TaqMan probe (Applied Biosystems, Foster, CA, USA) in an ABI 7900 real-time PCR system (Applied Biosystems).

Primer sequences were as follows: sense: 5’-AAG GGA GTC AGA AGA CAG ATG-3’; antisense: 5’-CAG GCA GTG AGG ATA CGA AC-3’. Cycling conditions were 5 minutes at 95°C, 45 cycles of 15 seconds at 95°C,1 minute at 60°C, and 30 seconds of cooling at 40°C.

Results were analyzed using real-time StatMineTMsoftware (Applied Biosystems). The accuracy of the genotyping assays was confirmed by repeat analysis of 15%of the samples. Concordance between the original and repeat genotype was 99%.

Statistical analysis

The distribution of genotypes according to the Hardy-Weinberg equilibrium was determined for both groups. Pearson’s chi-square test was used for differences in genotypic and allelic distribution between patients and healthy controls. OR and 95% CI per genotype and allele were estimated by applying unconditional logistic regression between patients with schizophrenia and healthy controls. Calculations were performed using SPSS for Windows, version 11.5 software (SPSS, Chicago, IL, USA). The level of statistical significance was determined at P ＜ 0.05.

Author contributions:Guangxin Wang and Zhaoyun Du had access to all data, and participated in data integrity and data analysis accuracy, study design, study supervision, and manuscript development. All authors participated in data collection.

Conflicts of interest:None declared.

Funding:The study was financially sponsored by the Scientific Foundation of Shandong Population and Family Planning, No.2006-7.

Ethical approval:The study was approved by the Ethics Committee of Jining Medical College, China.

[1]Wu M, Lu JC. Association of neuregulin-1 with schizophrenia.Neural Regen Res. 2008;3(2):229-232.

[2]Moorhead TW, Stanfield A, Spencer M, et al. Progressive temporal lobe grey matter loss in adolescents with schizotypal traits and mild intellectual impairment. Psychiatry Res. 2009;174(2):105-109.

[3]Aukes MF, Alizadeh BZ, Sitskoorn MM, et al. Finding suitable phenotypes for genetic studies of schizophrenia: heritability and segregation analysis. Biol Psychiatry. 2008;64(2):128-136.

[4]Kim Y, Zerwas S, Trace SE, et al. Schizophrenia genetics: where next? Schizophr Bull. 2011;37(3):456-463.

[5]Mantripragada KK, Carroll LS, Williams NM. Experimental approaches for identifying schizophrenia risk genes. Curr Top Behav Neurosci. 2010;4:587-610.

[6]Grymek K, Lukasiewicz S, aron-Góreckaa A, et al. Role of silent polymorphisms within the dopamine D1 receptor associated with schizophrenia on Dl-D2 receptor hetero-dimerization. Pharmacol Rep. 2009;61(6):1024-1033.

[7]Grandy DK, Zhou QY, Allele L, et al. A human D1 dopamine receptor gene is located on chromosome 5 at q35.1 and identifies an EcoRI RFLP. Am J Hum Genet. 1990;47(5):828-834.

[8]Rybakowski JK, Borkowska A, Czerski PM, et al. An association study of dopamine receptors polymorphisms and the Wisconsin Card Sorting Test in schizophrenia. J Neural Transm. 2005;112:1575-1582.

[9]Abi-Dargham A, Moore H. Prefrontal DA transmission at D1 receptors and the pathology of schizophrenia. Neuroscientist.2003;9:404-416.

[10]Ioannidis JP, Boffetta P, Little J, et al. Assessment of cumulative evidence on genetic associations: interim guidelines. Int J Epidemiol. 2008;37:133-135.

[11]Zhang C, Fang Y, Xie B, et al. No genetic association between dopamine D1 receptor gene and [early onset]schizophrenia.Psychiatry Res. 2010;177(3):350-353.

[12]Takeshi K, Nemoto T, Fumoto M, et al. Reduced prefrontal cortex activation during divergent thinking in schizophrenia: a multi-channel NIRS study. Prog Neuropsychopharmacol Biol Psychiatry. 2010;34(7):1327-1332.

[13]Foster A, Miller del D, Buckley P. Pharmacogenetics and schizophrenia. Clin Lab Med. 2010;30(4):975-993.

[14]Duan J, Sanders AR, Gejman PV. Genome-wide approaches to schizophrenia. Brain Res Bull. 2010;83(3-4):93-102.

[15]Nieratschker V, Nöthen MM, Rietschel M. New genetic findings in schizophrenia: is there still room for the dopamine hypothesis of schizophrenia? Front Behav Neurosci. 2010;4:23.

[16]Wu DX, Yin HF, Yan LR, et al. Brain activation regions in schizophrenia patients performing the game piece memory task.Neural Regen Res. 2009;4(2):98-105.

[17]Heinz A, Schlagenhauf F. Dopaminergic dysfunction in schizophrenia: salience attribution revisited. Schizophr Bull. 2010;36(3):472-485.

[18]Remington G, Agid O, Foussias G. Schizophrenia as a disorder of too little dopamine: implications for symptoms and treatment.Expert Rev Neurother. 2011;11(4):589-607.

[19]Allele NC, Bagade S, McQueen MB, et al. Systematic meta-analyses and field synopsis of genetic association studies in schizophrenia: the SzGene database. Nat Genet. 2008;40(7):827-834.

[20]Fung MM, Rana BK, Tang CM, et al. Dopamine D1 receptor(DRD1) genetic polymorphism: pleiotropic effects on heritable renal traits. Kidney Int. 2009;76(10):1070-1080.

[21]American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. 4thed. Washington DC: American Psychiatric Press. 1994.