Polymorphism of DTNBP 1 Gene P 1635 ( Rs 3213207 ) In Schizophrenic Javanese , Indonesia

Background: Many putative genes were predicted as risk factors of schizophrenia. One of these genes is DTNBP1 gene. One of single nucleotide polymorphism (SNPs) of DTNBP1 gene is P1635 (rs3213207). Objective: This study aimed to find out the association between DTNBP1 gene, P1635 (rs3213207), and risk of schizophrenia. Methods: DNA was isolated from blood of 25 schizophrenia patients and 25 healthy controls. DNA was applied to restriction fragment length polymorphism polymerase chain reaction (RFLP PCR). PCR products were digested by BSr1 enzyme. Results: The results show that there are 24 AA genotype (wild type) from 25 Javanese schizophrenia patients and 1 GG homozygote, while all 25 of controls are AA genotype (wild type). The p value of Fisher’s exact test is 0,500. Conclusion: There is no significant differences of genotype distribution of SNP P 1635(rs3213207) between schizophrenia patients and healthy controls in Javanese population


Introduction
Schizophrenia is a disease with mental deterioration. The symptoms of this disease include hallucinations, dilution, speech disorder, catatonic reflexes and others 1 . The prevalence of this disease approximately 1% 2,3 . The disorder causes a reduction in affective and social abilities. The most age with schizophrenia is 15-35 years old 4 . In 1995, the prevalence of schizophrenia in Indonesia (>15 years old) was approximately 3 in 1000 household 5 . Genetic factor thought to be one of predisposing factors of schizophrenia. Several genes were predicted as a risk factor for schizophrenia, including Catechol O methyltransferase (COMPT) gene 6 and Dystrobrevin binding protein 1 (DTNBP1) gene in schizophrenia Iranian population 7 . Dystrobrevin binding protein 1 is genes that function in the formation of dysbindin (Dystrobrevin-binding protein 1) 8 . DTNBP1 gene is located on chromosome 6p22.3. Several studies state that this gene increases the risk of schizophrenia. Dysbindin has molecular weight of 40 kD. This protein is bound to dystrobrevin (a complex glycoprotein dystrophin) 9 .
The DTNBP1 gene was allegedly affecting the incidence of schizophrenia. Variation in this gene affects the expression and processes in mRNA 10 . Research conducted by Weikert et al., 2004 showed that schizophrenics have low levels dysbindin 11 . The polymorphisms of DTNBP1 gene at p 1635 (located in intron 4) are predicted to affect the incidence of schizophrenia. An allele at p1635 increases the risk of schizophrenia, while G allele lowers the risk of schizophrenia in Iranian schizophrenia patients 12 .

Material and Methods
A total of 25 schizophrenia patients and 25 healthy controls were used as research subjects. DNA was isolated from blood of the subjects. DNA isolation was performed as follows: 20 mL of Proteinase K was put in a 1.5 mL tube then 200 μL of blood sample was added into the tube and mixed. 200 mL of buffer was added to the mixture and mix by vortexing for 15 seconds. The mixtures were incubated in a water bath at temperature of 56°C for 10 minutes followed by centrifugation. Supernatant was removed and 200 mL of absolute ethanol was added to the pellet followed by vortexing for 15 seconds. Sample was mixed and placed in a column and centrifuged at 8000 rpm for 1 minute. Column was removed and transferred to a new tube and 500 mL of buffer AW 1 (Qiagen kit) was added to the column. Sample was centrifuged at 8000 rpm for 1 minute and column was transferred into a new tube. 500 mL of buffer AW 2 (Qiagen kit) was added and then centrifuged at 13,000 rpm for 3 minutes. Column was transferred in a 1.5 mL new tube and 200 mL of buffer AE (Qiagen kit) was added to the column. This mixture was maintained for 1 minute at room temperature and then centrifuged at 8000 rpm for 1 minute. Polymerase chain reaction (PCR): PCR was performed by restriction fragment length polymorphism (RFLP) PCR. A total of 12,5 μL master mix, 6,5 μL dH 2 O, 2 μL forward primer: 5-GCAGACCATG-TATTTGAAAAGC-3, 2 μL reverse primer: 5-GCCAGTTTC-CTCAAAATTCC-3) and 2 μL DNA were run by PCR. The thermo cycling condition as follows: the initial denaturation was 94°C for 5 minutes, followed by 35 cycles at 95°C for 30 seconds, 54°C for 30 seconds and 72°C for 30 seconds. The final step was 72°C for 5 minutes and 12°C for 10 minutes. PCR products were digested by BSrI enzyme. The digestion products were analyzed by gel electrophoresis using 12% agarose gel.

Analyzes
The differences of genotype distribution of SNP P1635 between schizophrenia patients and healthy controls were analyzed by Fisher's exact test.  Based on Table 1, it can be seen that homozygous GG was found in one patient with schizophrenia and not found in the control (n = 25). The p value on Fisher's exact test is 0,500. Discussion DTNBP1 gene encodes dysbindin (dystrobrevin binding protein 1). This gene is widely expressed in the brain. This gene is thought to play an important role in cognitive function and memory 8 . The gene is located on 6p22.3 and thought to correlate with the incidence of schizophrenia 13

Conclusion
There is no significant difference in genotype distribution of SNP 1635 in schizophrenia patients and healthy controls in Javanese population (P> 0.05)