本文關(guān)鍵詞： X-性連鎖隱性視網(wǎng)膜色素變性 視網(wǎng)膜GTP酶調(diào)節(jié)因子基因 retinitis pigmentosa 2基因 突變檢測(cè)　出處：《安徽醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的分析兩個(gè)X-性連鎖視網(wǎng)膜色素變性(X-linked retinitis pigmentosa ,XLRP)家系的臨床表型特征。通過(guò)基因序列分析對(duì)XLRP候選基因進(jìn)行突變檢測(cè),尋找致病基因。 方法對(duì)兩個(gè)RP家系在獲得知情同意的前提下對(duì)家系成員進(jìn)行病史采集,眼部檢查、包括眼前節(jié)和視網(wǎng)膜功能和形態(tài)學(xué)檢查,繪制家系系譜,同時(shí)對(duì)家系成員抽取外周靜脈血,并提取DNA,為進(jìn)一步確保研究的科學(xué)性,在門診隨機(jī)抽取100名健康者的外周血,并抽取DNA,作為對(duì)照使用。應(yīng)用引物設(shè)計(jì)軟件Primer3設(shè)計(jì)候選基因RPGR、RP2以及ORF15的引物,采用聚合酶鏈反應(yīng)(PCR)技術(shù)擴(kuò)增目的片段,所有PCR產(chǎn)物送往上海生工生物工程技術(shù)服務(wù)有限公司純化后, ABI公司3170自動(dòng)測(cè)序儀上以相應(yīng)PCR引物進(jìn)行雙向直接測(cè)序,檢測(cè)RP2基因和RPGR基因的所有外顯子和內(nèi)含子交界處序列,包括RPGR基因突變熱區(qū)15號(hào)外顯子開(kāi)放閱讀框(ORF15)。測(cè)序結(jié)果采用DNAstar軟件進(jìn)行序列分析。 結(jié)果根據(jù)遺傳圖譜確認(rèn)此兩個(gè)RP家系為性連鎖隱性遺傳,測(cè)序分析發(fā)現(xiàn)家系1患者的RPGR基因ORF15區(qū)中有2種單個(gè)堿基的改變,但在100名正常人測(cè)序中,也發(fā)現(xiàn)約20%～30%人有這2個(gè)堿基改變,考慮為單核苷酸多態(tài)性。在NCBI-SNP數(shù)據(jù)庫(kù)進(jìn)行檢索分析,分別是為c.3396 C→T,(p.N1132N)和c.3430位G→A的改變(p.V1144I),RPGR和RP2基因中沒(méi)有檢測(cè)到突變。 結(jié)論對(duì)兩個(gè)XLRP家系進(jìn)行了候選基因篩查,RPGR和RP2基因,均未發(fā)現(xiàn)基因突變。但發(fā)現(xiàn)家系1在RPGR的突變熱點(diǎn)ORF15外顯子有2個(gè)堿基變異,第3396位堿基C→T和第3430位堿基的G→A的轉(zhuǎn)換, 3396位C→T堿基的置換并未改變所編碼的氨基酸;3430位G→A的轉(zhuǎn)換引起了氨基酸序列的改變,但由于在正常人中也檢測(cè)到該變異,故認(rèn)為該位點(diǎn)的變異是一種多態(tài)現(xiàn)象。此外,本研究排除了RPGR和RP2這兩個(gè)XLRP家系的致病基因,為進(jìn)一步尋找致病基因奠定了一個(gè)基礎(chǔ)。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R774.1

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