精神分裂症候選基因的SNP分析與統計

Statistically Analyzing Correlations between Schizophrenia and Single Nucleotide Polymorphisms in Candidate Genes

施佳伶(Chia-Ling Shih)

指導教授 ： 王淑紅

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2013年)

https://doi.org/10.6834/CSMU.2013.00142

摘要

精神分裂症是一種複雜的精神疾病，在全球的盛行率約百分之一。雖然關於精神分裂症致病原因有許多假說，但我們主要探討多巴胺跟麩氨酸假說中相關基因與精神分裂症關聯性。我們利用單一核苷酸多態性（single nucleotide polymorphisms, SNPs）分析多巴胺假說中的DRD2、DRD3、 BDNF和COMT基因，和麩氨酸假說中的GRIN1、NRG1、DAOA 和DTNBP1基因。我們分析這些候選基因的單一核苷酸多型性與精神分裂症的相關性。
我們把實驗分成三個組別：用藥控制良好（第一型）和用藥控制不良（第二型）的精神分裂症病患，與正常人。我們分析8個基因共13個SNPs，當中屬於多巴胺假說分的DRD2基因（SNP-rs6277、SNP-rs1801028和SNP-rs6275）、DRD3基因（SNP-rs6280）、BDNF基因（SNP-rs6265）和COMT基因（SNP-rs4680）；屬於麩氨酸假說的GRIN1基因（SNP-rs6293）、NRG1基因（SNP-rs3924999和SNP-rs10503929）、DAOA基因（SNP-rs2391191）和DTNBP1基因（SNP-rs2619539、SNP-rs146937431和SNP-rs3213207）。分別設計引子來進行聚合酶鏈鎖反應（PCR），擴增各SNP附近序列。首先確認是否有插入或缺失突變，之後再進行序列分析，確認個別SNP是否有點突變和附近序列的正確性，並比較這些突變是否與精神分裂症有統計意義的關聯，我們也分析台灣人的對偶基因頻率與NCBI資料庫上的是否有差異。
雖然我們共分析13個在其他人種被證明與精神分裂症相關的SNPs，但是在台灣人中只有兩組SNPs的結果顯示與精神分裂症有相關性。我們的實驗結果發現BDNF基因的SNP-rs6265在女性與第一型精神分裂症有相關性(p＝0.044)，且DTNBP1基因的SNP-rs3213207與精神分裂症有關聯性(p＜0.001)，而其餘11組SNPs皆與精神分裂症無關聯性。此外，在對偶基因頻率方面有六個SNPs（SNP-rs6280、SNP-rs6265、SNP-rs2619539、SNP-rs3213207、SNP-rs3924999和SNP-rs2391191）在台灣人的結果與NCBI資料庫上的不相同，我們推測此差異可能主要是人類種族的差異所造成。對DTNBP1基因的SNP-rs2391191，我們進一步分析其mRNA表現量在不同組別與SNP的差異性，發現在第一型精神分裂症病患與正常人的DTNBP1 mRNA表現量在不同對偶基因上的趨勢是一致的，AG異型合子的mRNA表現量比AA同型合子的表現量低，這樣的降低趨勢可能是造成罹患精神分裂症與病患用藥控制良好的原因。
本研究結果證明BDNF和DTNBP1的各一個SNP之基因多態性與台灣的精神分裂症有關聯性，rs6265和rs3213207這兩組SNPs變異，可能在我們台灣人族群是造成罹患精神分裂症的因子之一。

關鍵字

精神分裂症 ； BDNF基因 ； DTNBP1基因 ； 單一核苷酸多態性

並列摘要

Schizophrenia is a complex psychiatric disorder which affects approximately 1% of human population worldwide. Although there are several different hypothesis based on facets of the disease, we majorly investigate the dopamine and glutamate hypothesis of candidate genes associated with schizophrenia. We analyzed the single nucleotide polymorphisms (SNPs) in DRD2, DRD3, BDNF and COMT genes for dopamine hypothesis, and SNPs in GRIN1, NRG1, DAOA and DTNBP1 genes for glutamate hypothesis. We tested the correlations of SNPs in these candidate genes with schizophrenia. We included three experimental groups: the effective drug treatment (type I) and ineffective drug treatment (type II) of schizophrenial patients, and normal persons without any known mental illness inheritance (control). These eight genes of thirteen SNPs including rs6277, rs1801028, rs6275, rs6280, rs6265, rs4680, rs6293, rs3924999, rs10503929, rs2391191, rs2619539, rs146937431 and rs3213207 were analyzed. Primers were designed for polymerase chain reaction (PCR) amplifications of sequences around SNPs, respectively. The insertions or deletions around these SNPs were firstly obtained. Each PCR product with correct size was then sequenced, and result was analyzed to decipher polymorphisms. The correlations of schizophrenia and polymorphisms were statistically analyzed. The allele frequencies between these samples and information in the National Center for Biotechnology Information (NCBI) databank were compared. Although these thirteen SNPs that show significant correlations with schizophrenia in other human populations, only two SNPs are significantly associated with schizophrenia in Taiwanese. Results of individual allele frequency associations show that SNP-rs6265 of BDNF gene (p=0.048) in female type I schizophrenial patients and SNP-rs3213207 of DTNBP1 gene (p＜0.001) in male and female type I schizophrenial patients are significantly associated with schizophrenia. Furthermore, the allele frequencies of six SNPs (rs6280, rs6265, rs2619539, rs3213207, rs3924999 and rs2391191) in Taiwanese are different from results shown in the NCBI databank. Ours data conjectures that these differences of SNPs might be caused by ethnic. Further, the expression levels of DTNBP1 mRNA for rs3213207 in various samples of different groups were analyzed. We found that DTNBP1 mRNA expression levels in type I schizophrenial patients and normal persons trend to be lower in the AG heterozygote samples compared with AA homozygous samples. This decrease may explain the why type I schizophrenia patients show effective drug treatment. These results show that the gene polymorphisms of BDNF and DTNBP1 are associated with schizophrenia in Taiwan. Variations of SNPs in rs6265 and rs3213207 may be factors causing schizophrenia in Taiwan.

並列關鍵字

Schizophrenia ； BDNF gene ； DTNBP1 gene ； Single nucleotide polymorphisms

參考文獻

1. Huang XY, Sun FK, Yen WJ, Fu CM. The coping experiences of carers who live with someone who has schizophrenia. Journal of clinical nursing. 2008;17(6):817-26.

2. Petronis A, Paterson AD, Kennedy JL. Schizophrenia: an epigenetic puzzle? Schizophrenia bulletin. 1999;25(4):639-55.

3. Kety SS, Rosenthal D, Wender PH, Schulsinger F. The types and prevalence of mental illness in the biological and adoptive families of adopted schizophrenics. J Psychiatr Res. 1968;6(suppl 1):345-62.

4. Tsuang MT. Recent advances in genetic research on schizophrenia. Journal of biomedical science. 1998;5(1):28-30.

6. Karreman M, Moghaddam B. The prefrontal cortex regulates the basal release of dopamine in the limbic striatum: an effect mediated by ventral tegmental area. Journal of neurochemistry. 1996;66(2):589-98.