序列捕獲高通量測序技術(shù)建立citrin缺乏癥基因檢測平臺及其應(yīng)用
發(fā)布時間：2019-03-06 17:11
【摘要】：Citrin缺乏癥是因SLC25A13基因突變導(dǎo)致其編碼的Citrin蛋白功能缺失從而出現(xiàn)一系列的臨床表現(xiàn)和生化檢測指標異常的常染色體隱性遺傳疾病。近年來研究發(fā)現(xiàn)該遺傳病呈現(xiàn)世界性分布,但以東亞地區(qū)多發(fā),包括我國在內(nèi)。SLC25A13基因突變分析檢測是目前確診Citrin缺乏癥的主要依據(jù)。當(dāng)前SLC25A13基因突變分析的方法主要是經(jīng)典的Sanger測序技術(shù)和聚合酶鏈反應(yīng)-限制性片段長度多態(tài)性技術(shù)(PCR-RFLP)等,而這些技術(shù)都有其缺點:Sanger測序技術(shù)操作繁瑣、通量低等;PCR-RFLP技術(shù)則不能發(fā)現(xiàn)新突變等。本研究希望建立基于高通量測序技術(shù)與目標序列捕獲技術(shù)的SLC25A13基因檢測新型平臺。本研究中高通量測序技術(shù)與目標序列捕獲技術(shù)結(jié)合的基因檢測平臺以炎黃YH標準基因組DNA測試整個試驗檢測流程并確定平臺的數(shù)據(jù)過濾方法。其后,連續(xù)檢測100個正常人基因組DNA樣品和2個citrin缺乏癥患者的基因組DNA樣品。為了篩選致病未報道的新的候選突變,利用多款預(yù)測軟件(如SIFT、Polyphen、GERP、LRT等)對這些突變進行有害性和保守型預(yù)測,最終鑒定導(dǎo)致疾病發(fā)生的相關(guān)基因及突變。其結(jié)果建立了100個正常中國SLC25A13和ASS1基因變異頻率表,以及在2名citrin缺乏癥患者的SLC25A13基因上檢出致病突變:c.851_854del GTAT/c.1177+1GA與c.754GA/c.1177+1GA。其中c.754GA(p.Glu252 Lys)之前未見中外報道,對其進行預(yù)測分析為其位置具有高度保守性和該突變具有有害性或致病性。通過經(jīng)典Sanger測序?qū)颊呒捌浼覍龠M行驗證,其結(jié)果為本研究中高通量測序結(jié)果與Sanger測序結(jié)果相一致。同時,也說明了患者分別從其父母雙方遺傳得到一個致病突變,使得患者在SLC25A13基因存在復(fù)合雜合突變,這與citrin缺乏癥的常染色體隱性遺傳方式相一致。本研究說明了由高通量測序技術(shù)與目標序列捕獲技術(shù)所組成的基因檢測平臺應(yīng)是一種為診斷某些明確發(fā)病機制的遺傳病而檢測其基因的有效方法,包括本研究中的citrin缺乏癥(瓜氨酸血癥II型)。同時,該平臺對其他遺傳病或基因缺陷病都有著重要的應(yīng)用價值和研究意義。
[Abstract]:Citrin deficiency is an autosomal recessive genetic disease caused by mutation of SLC25A13 gene resulting in a series of autosomal recessive genetic diseases, which lead to a series of clinical manifestations and abnormal biochemical markers. In recent years, it has been found that this genetic disease has a worldwide distribution, but it is common in East Asia, including China. Analysis and detection of SLC25A13 gene mutation is the main basis for the diagnosis of Citrin deficiency at present. The methods of SLC25A13 gene mutation analysis are classical Sanger sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and so on. These techniques have their disadvantages: Sanger sequencing is tedious and its throughput is low. PCR-RFLP technique can not find new mutations and so on. In this study, we hope to establish a new platform for SLC25A13 gene detection based on high-throughput sequencing and target sequence capture. In this study, a gene detection platform combined with high-throughput sequencing technology and target sequence capture technology was used to test the whole experimental detection flow and determine the data filtering method of the platform with the standard genomic DNA of Yanhuang YH. Then, the genomic DNA samples from 100 normal subjects and 2 citrin deficiency patients were detected continuously. In order to screen new candidate mutations that have not been reported, several prediction software (such as SIFT,Polyphen,GERP,LRT, etc.) are used to predict these mutations in order to identify the related genes and mutations that lead to disease occurrence. The results showed that the frequency tables of SLC25A13 and ASS1 gene mutation were established in 100 normal Chinese, and the pathogenic mutations were detected in SLC25A13 gene of 2 citrin deficiency patients: c.851_854del GTAT/c.1177 1GA and c.754GA/c.1177 1GA. Among them, c.754GA (p.Glu252 Lys) has not been reported at home and abroad before. The prediction analysis shows that its position is highly conservative and the mutation is noxious or pathogenicity. The patients and their families were verified by classical Sanger sequencing. The results showed that the results of high throughput sequencing in this study were consistent with the results of Sanger sequencing. At the same time, it also showed that the patient got a disease-causing mutation from both parents, which resulted in a complex heterozygous mutation in the SLC25A13 gene, which was consistent with the autosomal recessive inheritance pattern of citrin deficiency. This study shows that the gene detection platform composed of high-throughput sequencing and target sequence capture techniques should be an effective method for the diagnosis of certain genetic diseases with clear pathogenesis. Including citrin deficiency in this study (citrullinemia type II). At the same time, the platform has important application value and research significance to other genetic diseases or gene deficiency diseases.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R596;Q78
本文編號：2435730
[Abstract]:Citrin deficiency is an autosomal recessive genetic disease caused by mutation of SLC25A13 gene resulting in a series of autosomal recessive genetic diseases, which lead to a series of clinical manifestations and abnormal biochemical markers. In recent years, it has been found that this genetic disease has a worldwide distribution, but it is common in East Asia, including China. Analysis and detection of SLC25A13 gene mutation is the main basis for the diagnosis of Citrin deficiency at present. The methods of SLC25A13 gene mutation analysis are classical Sanger sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and so on. These techniques have their disadvantages: Sanger sequencing is tedious and its throughput is low. PCR-RFLP technique can not find new mutations and so on. In this study, we hope to establish a new platform for SLC25A13 gene detection based on high-throughput sequencing and target sequence capture. In this study, a gene detection platform combined with high-throughput sequencing technology and target sequence capture technology was used to test the whole experimental detection flow and determine the data filtering method of the platform with the standard genomic DNA of Yanhuang YH. Then, the genomic DNA samples from 100 normal subjects and 2 citrin deficiency patients were detected continuously. In order to screen new candidate mutations that have not been reported, several prediction software (such as SIFT,Polyphen,GERP,LRT, etc.) are used to predict these mutations in order to identify the related genes and mutations that lead to disease occurrence. The results showed that the frequency tables of SLC25A13 and ASS1 gene mutation were established in 100 normal Chinese, and the pathogenic mutations were detected in SLC25A13 gene of 2 citrin deficiency patients: c.851_854del GTAT/c.1177 1GA and c.754GA/c.1177 1GA. Among them, c.754GA (p.Glu252 Lys) has not been reported at home and abroad before. The prediction analysis shows that its position is highly conservative and the mutation is noxious or pathogenicity. The patients and their families were verified by classical Sanger sequencing. The results showed that the results of high throughput sequencing in this study were consistent with the results of Sanger sequencing. At the same time, it also showed that the patient got a disease-causing mutation from both parents, which resulted in a complex heterozygous mutation in the SLC25A13 gene, which was consistent with the autosomal recessive inheritance pattern of citrin deficiency. This study shows that the gene detection platform composed of high-throughput sequencing and target sequence capture techniques should be an effective method for the diagnosis of certain genetic diseases with clear pathogenesis. Including citrin deficiency in this study (citrullinemia type II). At the same time, the platform has important application value and research significance to other genetic diseases or gene deficiency diseases.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R596;Q78
【參考文獻】
相關(guān)期刊論文 前2條
1 溫鵬強;王國兵;陳占玲;崔冬;袁泉;宋萍;陳淑麗;廖建湘;李成榮;;Citrin缺陷導(dǎo)致的新生兒肝內(nèi)膽汁淤積癥SLC25A13基因分析[J];中國當(dāng)代兒科雜志;2011年04期
2 佐伯武賴;小林圭子;張春花;王建設(shè);;Citrin缺陷病及其研究帶來的啟示[J];中國循證兒科雜志;2010年06期
，本文編號：2435730
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