Mutation Screening and Translocation Detection of DISC1 Gene in Schizophrenia
试验速览
- 阶段
- 不适用
- 状态
- 已完成
- 入组人数
- 300
研究概览
简要总结
Several chromosomal loci obtained from genetic linkage studies have been reported of relating to schizophrenia. These areas include of 1q32-41, 6p24-21, 8p22-21, 15q13-14, and 22q11-12. The names of these genes located in these loci have not been identified, nor have the function and the relationship to the disease. Our research team using genetic linkage studies has found a strong linkage (NPL Z score = 2.18, p=0.01) between the D1S251 marker and schizophrenia disease. This marker is about 4 kb away from DISC1 (disrupted in schizophrenia gene 1) gene. In a Scottish family, a balanced translocation [t(1;11) (q42.1;q14.3)] has cosegregated inside the schizophrenia affected members of the family (LOD score =6.0). The breakpoint of the translocation is located at the intron area between exon 8 and exon 9 of DISC1 gene. This translocation disrupted the gene and caused its malfunction. A large molecular genetic study study recently in Finland has also demonstrated strong linkage evidence (Zmax=3.21) between the DIS2709 marker, located among exon 4 and exon 5 of DISC1 gene, and schizophrenia in a. All these findings have indicated that DISC1 gene is a potential positional candidate gene and worth for further study.
The main purposes of this proposal include: (1) To evaluate the incidence rate of the balanced translocation between the chromosome 1q42.1 and 11q14.3 in approximately 500 schizophrenic patients in Taiwan. Furthermore, we will compare the clinical symptoms, illness course, and family genetic model to examine if any particular characters coexist with the translocation. (2) To search for the genetic polymorphisms in DISC1 gene area, where the thirteen exons, the promoter regions (1 kb upstream the start codon), and the breakpoint area (1 kb of both upstream and downstream area) will be analyzed by the method of denaturing high performance liquid chromatography (DHPLC). Case-control association study will be performed further in each 200 schizophrenic patients and normal controls to evaluate the relationship between the disease and the clinical characteristics.
This proposal is quite feasible and prospective with the following reasons. (1) All the DNA samples and the clinical data have been collected and evaluated completely for further analysis. (2) Our research team has built up an integrated andreliable molecular genetic laboratory. All the facilities necessary for this study (DHPLC) had been setup with standard operating protocols and working routinely. (3) DISC1 gene has strong linkage evidence with schizophrenia in Taiwanese sample. The high prior probability of DISC1 gene as a positional candidate gene increases the successfulness of association study. . (4) Once the relationship among balance translocation, genetic polymorphism and the schizophrenia have established, further functional study will be evaluated to understand the possible mechanisms involved in the disease.
详细描述
The grant proposal has two major research goals. The first goal will be constructing an E.coli plasmid carried with an approximately 1.4 kb of DNA sequences from each 700 bps of the chromosome 1q42.1 and 11q14.3 around the breakpoint. The second goal will be establishing the denaturing high performance liquid chromatography (DHPLC) methods to screen the mutations or polymorphisms for all the exons of DISC1 gene, the 1 kb at the promoter region, and the sequences from 1 kb at the upstream and downstream of the breakpoint.
Translocation (1q42.1;11q14.3)-Carried Plasmid DNA Construction
Human genomic DNA isolation All human genomic DNA will be used from these subjects collected in the past, or subjects collected specific for the plasmid construction study with informed consents. For the plasmid construction, mononuclear leukocytes will be isolated by a modification of the method of Böyum (1968). Whole blood with EDTA as anticoagulant will be taken by venipuncture from healthy volunteers. The collected blood will be diluted with an equal volume of phosphate buffered saline (PBS) and layered onto Histopaque-1077. After separation by centrifugation at 400xg for 40 min, the mononuclear leukocyte layer will be removed and washed three times with PBS by centrifuging cells at 400xg for 10 min. The freshly isolated cells will be further applied for genomic DNA isolation according to standard phenol/chloroform extraction procedures (Sambrook et al. 1989).
PCR amplification of 1q42.1 and 11q14.3 DNA sequences The amplification will be performed with AmpliTaq Gold DNA polymerase in a 50 ul reaction volume. The reaction containing 50 ng DNA , 1 U of enzyme, 300 ng of each primer, 200 mM of each dNTP, 1.5 mM MgCl2, 50 mM KCl and 10 mM Tris-HCl, pH8.3. All reaction will be performed with an initial denaturing step of 5 min at 95℃ followed by 35 cycles of a denaturing step at 94℃ for 30 sec, an annealing step of 1 min at a temperature appropriate for the primers used, and a synthesis step at 72℃ for 10 min. The primer sequence for the 738 bps of upstream breakpoint at 1q42.1 will be designed with an EcoRI cutting site at the breakpoint end. The primer sequence for the 719 bps of downstream breakpoint at 11q14.3 will also be designed with an EcoRI cutting site at the breakpoint end.
Ligating the two PCR DNA fragments and purification by gel electrophoresis The PCR product of both 1q42.1 and 11q14.3 will be reacted with EcoRI separately to reveal the cohesive ligation sites. Two of the DNA fragments, around 700 bps each, will be ligated by T4 DNA ligase following the protocol provided from the InsT/AcloneTM cloning kit (MBI Fermentas, U.S.A.). The ligated 1.4 kb of DNA fragment will be re-amplified by above PCR procedure, purified and recovered from agarose gel by electroelution and extraction with organic solvents (Sambrook et al. 1989).
研究设计
- 研究类型
- Observational
- 观察模型
- Defined Population
- 时间视角
- Other
入排标准
- 年龄范围
- 18 Years 至 65 Years（Adult, Older Adult）
- 性别
- All
- 接受健康志愿者
- 是
入选标准
- •Schizophrenia
- •Chinese Han People
排除标准
- 未提供