DTNBP1 Gene Is Associated with Some Symptom Factors of Schizophrenia in Chinese Han Nationality△

Yu-hui Sun, Yan Shen, and Qi Xu

National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China

SCHIZOPHRENIA is a severe form of mental illness affecting about 1% of the adult population. It is often clinically described in terms of positive and negative symptoms, and cognitive deficits, which are assessed with Positive and Negative Syndrome Scale (PANSS) in clinical practice.1

DTNBP1 gene (6p22.3) was first reported to be associated with schizophrenia in 270 Irish high-density pedigrees.2In the next few years, DTNBP1 gene was recognized as one of the hottest schizophrenia susceptibility genes based on case-control and family-based association studies in different populations.3-10Yet consistent polymorphisms or haplotypes were not identified until now. There are also evidences showing that dysbindin is involved in the pathology of schizophrenia at both mRNA and protein levels.11-14However, some genetic results previously reported showed no association between DTNBP1 gene and schizophrenia.15-18In some other studies, that gene of interest was shown to be associated with negative symptoms of schizophrenia, cognitive deficits, working memory, and some other phenotypes.19-26

In the present study, we selected four single nucleotide polymorphisms (SNPs) (rs742106, rs909706, rs1011313, and rs2619539) in DTNBP1 gene previously reported associated with schizophrenia6,10,24,27,28to test whether these polymorphisms were associated with some symptom factors of that mental illness.

PATIENTS AND METHODS

Patients

A total of 284 unrelated schizophrenia patients (131 males and 154 females, aged 31.9 ± 10.9 years) of Han nationality were recruited by Department of Psychiatry, the First Hospital of Shanxi Medical University, from December 2004 to January 2009. These patients were diagnosed with schizophrenia by at least two consultant psychia- trists in a clinical interview based on the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) (American Psychiatric Association, 1994). Patients were excluded from this study if they suffered from any organic brain disorders, mental retardation, severe head trauma, or demonstrate psychotic symptoms due to medical conditions or treatments. The included patients all pro- vided written informed consents to blood sample collection for genetic analysis. The protocol of this study was examined and approved by the Ethics Committee of Chinese Academy of Medical Sciences & Peking Union Medical College.

Clinical assessment of symptoms

For all the included patients, positive and negative symptoms were assessed according to the PANSS. The severity of each symptom was scored with a 7-grade scale (1: absent； 2: minimal； 3: mild； 4: moderate； 5: moderately severe； 6: severe； 7: extreme).

Genotyping of SNPs

Four SNPs present in the DTNBP1 gene were selected in the present study to be genotyped. Among those SNPs, rs909706 is located in intron 1, rs1011313 in intron 4, rs2619539 in intron 5, and rs742106 in intron 9.6,10,24,27,28

Genomic DNA was extracted from peripheral blood leukocytes following the standard phenol-chloroform procedure. The primers used for polymerase chain reaction (PCR) amplification were designed by the Primer Premier 5.0 software (Table 1). The reaction volume of PCR amplification was 25 μL, containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.001% (w/v) gelatin, 200 μmol/L of deoxyribonucleoside triphosphates (dNTPs), 0.4 μmol/L of the primers, 1.0 unit of Taq DNA polymerase (Tiangen, Beijing, China), and 50 ng of the extracted genomic DNA. The conditions for thermal cycling included an initial denaturation at 94°C for 5 minutes, 35 cycles at 94°C for 30 seconds, at 50-56°C for 30 seconds, and at 72°C for 30 seconds, followed by a final elongation at 72°C for 10 minutes on GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA, USA). The PCR products were purified and then sequenced bidirectionally with an ABI 3700 DNA sequencer (Applied Biosystems).

Statistical analysis

Hardy-Weinberg equilibrium for the genotypic distributions of each SNP was tested with the chi-square (χ2) goodness-of-fit test. Pair-wise linkage disequilibrium among these four SNPs was defined by the four-gamete rule using Haploview 4.2. The UNPHASED program (version 3.0.12) was applied to analyze genotype data.29The additive genetic value, which represents the difference in expected trait due to the allele compared with the reference, was estimated for determining the association between scored positive/negative symptoms and tested SNPs. The statistical significance level was set at 0.05 and adjusted by 10 000 permutations.

RESULTS

The χ2goodness-of-fit test showed that the genotypic distributions of the four studied SNPs were all in Hardy- Weinberg equilibrium in the schizophrenia patients (Table 2).

According to the plotting by Haploview 4.2, rs1011313 and rs2619539 were in one lingkage disequilibrium block in our samples, while the other two were not.

The quantitative trait test showed allelic association of rs909706 with the excitement symptom of schizophrenia (χ2=7.352, P＜0.05, adjusted by 10 000 permutations), and allele G with higher additive values than allele A (Table 3). In addition, rs2619539 was shown to be associated with lack of spontaneity and flow of conversation (χ2=9.151, P＜0.05, adjusted by 10 000 permutations), and the genotype C/G with a higher additive values than the other genotypes (Table 4).

Table 1. Polymerase chain reaction primers for genotyping of DTNBP1 SNPs

Table 2. Hardy-Weinberg equilibrium test of the four SNPs in schizophrenia patients

Table 3. Allelic association of four SNPs and excitement

Table 4. Genotypic association of four SNPs and lack of spontaneity and flow of conversation

We also carried out quantitative trait tests for other symptoms covered in PANSS, yet no associations were found between those symptoms and the studied SNPs.

DISCUSSION

Schizophrenia is a genetically complex disorder with an unknown pathophysiology. It is suggested that to study its specific symptoms or clinical phenotypes could help to establish a better understanding of this disorder.30The PANSS assessing the positive and negative symptoms of schizophrenia includes positive scales (delusions, conceptual disorganization, hallucinatory behavior, excitement, grandiosity, suspiciousness/persecution, and hostility), negative scales (blunted affect, emotional withdrawal, poor rapport, passive/apathetic social withdrawal, difficulty in abstract thinking, lack of spontaneity and flow of conversation, and stereotyped thinking), and general psychopathology scale (somatic concern, anxiety, guilt feelings, tension, mannerisms and posturing, depression, motor retardation, uncooperativeness, unusual thought content, disorientation, poor attention, lack of judgment and insight, disturbance of volition, poor impulse control, preoccupation, and active social avoidance).1

rs909706 and rs2619539, the two SNPs found in this study to be related with some symptoms of schizophrenia, are both intronic polymorphisms. rs909706 was found to contribute to schizophrenia,10and associated with change of patients' response to anti-psychotic agent haloperidol.28And rs2619539 was associated with methamphetamine psychosis,31less severe manic-type symptoms in psychosis,32and cognitive functions.23

Complementary to those previous findings, the quantitative trait test in our study showed association of some SNPs in DTNBP1 gene with some symptom factors of schizophrenia. In specific, rs909706 was associated with excitement, and rs2619539 with lack of spontaneity and flow of conversation. Their locations in different linkage disequilibrium blocks may explain the fact that those two symptoms belong to positive and negative scales respectively.

Our study revealed a possible association between DTNBP1 and the symptoms of schizophrenia, which perhaps could partly explain the relationship between DTNBP1 variations and that mental illness. Further investigation in large samples and different populations is still needed for clarifying the mechanisms of how the DTNBP1 gene affects the symptoms of schizophrenia.

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