X-性连锁隐性遗传视网膜色素变性致病基因的突变检测

发布时间：2018-02-13 16:20

本文关键词： X-性连锁隐性视网膜色素变性视网膜GTP酶调节因子基因 retinitis pigmentosa 2基因突变检测　出处：《安徽医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的分析两个X-性连锁视网膜色素变性(X-linked retinitis pigmentosa ,XLRP)家系的临床表型特征。通过基因序列分析对XLRP候选基因进行突变检测,寻找致病基因。方法对两个RP家系在获得知情同意的前提下对家系成员进行病史采集,眼部检查、包括眼前节和视网膜功能和形态学检查,绘制家系系谱,同时对家系成员抽取外周静脉血,并提取DNA,为进一步确保研究的科学性,在门诊随机抽取100名健康者的外周血,并抽取DNA,作为对照使用。应用引物设计软件Primer3设计候选基因RPGR、RP2以及ORF15的引物,采用聚合酶链反应(PCR)技术扩增目的片段,所有PCR产物送往上海生工生物工程技术服务有限公司纯化后, ABI公司3170自动测序仪上以相应PCR引物进行双向直接测序,检测RP2基因和RPGR基因的所有外显子和内含子交界处序列,包括RPGR基因突变热区15号外显子开放阅读框(ORF15)。测序结果采用DNAstar软件进行序列分析。结果根据遗传图谱确认此两个RP家系为性连锁隐性遗传,测序分析发现家系1患者的RPGR基因ORF15区中有2种单个碱基的改变,但在100名正常人测序中,也发现约20%～30%人有这2个碱基改变,考虑为单核苷酸多态性。在NCBI-SNP数据库进行检索分析,分别是为c.3396 C→T,(p.N1132N)和c.3430位G→A的改变(p.V1144I),RPGR和RP2基因中没有检测到突变。结论对两个XLRP家系进行了候选基因筛查,RPGR和RP2基因,均未发现基因突变。但发现家系1在RPGR的突变热点ORF15外显子有2个碱基变异,第3396位碱基C→T和第3430位碱基的G→A的转换, 3396位C→T碱基的置换并未改变所编码的氨基酸;3430位G→A的转换引起了氨基酸序列的改变,但由于在正常人中也检测到该变异,故认为该位点的变异是一种多态现象。此外,本研究排除了RPGR和RP2这两个XLRP家系的致病基因,为进一步寻找致病基因奠定了一个基础。
[Abstract]:Objective to analyze the clinical phenotypic characteristics of two families of X-linked retinitis pigmentosa (pigmentosa) with X- linked retinitis pigmentosa, and to detect the mutation of XLRP candidate genes by gene sequence analysis. Methods two RP families were collected with informed consent, eye examination, including anterior segment and retinal function and morphology, pedigree was drawn, and peripheral venous blood was drawn from the members of the family. In order to further ensure the scientific nature of the study, the peripheral blood samples of 100 healthy people were randomly selected from outpatients and used as control. The primer design software Primer3 was used to design the primers for candidate gene RPG RN RP2 and ORF15. Polymerase chain reaction (PCR) technique was used to amplify the target fragment. All the PCR products were sent to Shanghai Bioengineering Technology Services Co., Ltd. After purification, the ABI 3170 automatic sequencing instrument was used to carry out bidirectional direct sequencing with corresponding PCR primers. All the exons and introns of RP2 gene and RPGR gene were sequenced, including the open reading frame of exon 15 in the hot region of RPGR gene mutation. The sequencing results were analyzed by DNAstar software. Results according to the genetic map, the two RP families were identified as sex-linked recessive inheritance. Sequencing analysis showed that there were two kinds of single base changes in the ORF15 region of RPGR gene in family 1 patients, but in 100 normal persons. It was also found that about 20% of the population had these two base changes and considered single nucleotide polymorphisms. 鈫扵U p. N1132N) and c. 3430 G. 鈫扤o mutations were detected in the RPGR and RP2 genes. Conclusion two XLRP families were screened for candidate genes, and no mutation was found in either of them. However, it was found that family 1 had two base mutations in the ORF15 exon of RPGR mutation hot spot, the 3396 base. 鈫扜 of T and 3430 bases. 鈫扐 conversion, 3396 bits C. 鈫扵he substitution of the T base did not change the encoded amino acid sequence at position 3430 G. 鈫扵he change of A caused the change of amino acid sequence, but because the mutation was also detected in normal people, the mutation was considered to be a polymorphic phenomenon. In addition, this study excluded the pathogenic genes of RPGR and RP2. It lays a foundation for further searching for pathogenic genes.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.1

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