序列捕获高通量测序技术建立citrin缺乏症基因检测平台及其应用
发布时间：2019-03-06 17:11
【摘要】：Citrin缺乏症是因SLC25A13基因突变导致其编码的Citrin蛋白功能缺失从而出现一系列的临床表现和生化检测指标异常的常染色体隐性遗传疾病。近年来研究发现该遗传病呈现世界性分布,但以东亚地区多发,包括我国在内。SLC25A13基因突变分析检测是目前确诊Citrin缺乏症的主要依据。当前SLC25A13基因突变分析的方法主要是经典的Sanger测序技术和聚合酶链反应-限制性片段长度多态性技术(PCR-RFLP)等,而这些技术都有其缺点:Sanger测序技术操作繁琐、通量低等;PCR-RFLP技术则不能发现新突变等。本研究希望建立基于高通量测序技术与目标序列捕获技术的SLC25A13基因检测新型平台。本研究中高通量测序技术与目标序列捕获技术结合的基因检测平台以炎黄YH标准基因组DNA测试整个试验检测流程并确定平台的数据过滤方法。其后,连续检测100个正常人基因组DNA样品和2个citrin缺乏症患者的基因组DNA样品。为了筛选致病未报道的新的候选突变,利用多款预测软件(如SIFT、Polyphen、GERP、LRT等)对这些突变进行有害性和保守型预测,最终鉴定导致疾病发生的相关基因及突变。其结果建立了100个正常中国SLC25A13和ASS1基因变异频率表,以及在2名citrin缺乏症患者的SLC25A13基因上检出致病突变:c.851_854del GTAT/c.1177+1GA与c.754GA/c.1177+1GA。其中c.754GA(p.Glu252 Lys)之前未见中外报道,对其进行预测分析为其位置具有高度保守性和该突变具有有害性或致病性。通过经典Sanger测序对患者及其家属进行验证,其结果为本研究中高通量测序结果与Sanger测序结果相一致。同时,也说明了患者分别从其父母双方遗传得到一个致病突变,使得患者在SLC25A13基因存在复合杂合突变,这与citrin缺乏症的常染色体隐性遗传方式相一致。本研究说明了由高通量测序技术与目标序列捕获技术所组成的基因检测平台应是一种为诊断某些明确发病机制的遗传病而检测其基因的有效方法,包括本研究中的citrin缺乏症(瓜氨酸血症II型)。同时,该平台对其他遗传病或基因缺陷病都有着重要的应用价值和研究意义。
[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.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R596;Q78
本文编号：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.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R596;Q78
【参考文献】
相关期刊论文 前2条
1 温鹏强;王国兵;陈占玲;崔冬;袁泉;宋萍;陈淑丽;廖建湘;李成荣;;Citrin缺陷导致的新生儿肝内胆汁淤积症SLC25A13基因分析[J];中国当代儿科杂志;2011年04期
2 佐伯武赖;小林圭子;张春花;王建设;;Citrin缺陷病及其研究带来的启示[J];中国循证儿科杂志;2010年06期
，本文编号：2435730
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