|Alternative Title||Identification and functional study of pathogenic mutations in single gene diseases of the nervous system -a case study of metachromatic leukodystrophy|
本研究主要结果如下。1)本研究通过在一个具有常染色体隐性遗传模式的家系中，利用全外显子测序的方法鉴定到了先证者的疑似的致病位点ARSANM_ 000487.S:c.925G>Ap.E309K，该位点位于与异染性脑白质营养不良疾病相关的ARSA基因上，经过文献回顾发现，在以前的研究中，ARSA c.925G(c.925G>A, c.925G>T, c.925G>C)都被报道过可导致晚婴期异染性脑白质营养不良。2)基于该致病位点缺乏足够的生物学功能研究的现状，本研究通过构建突变细胞模型，使用系统生物学的方法如关键基因和蛋白一蛋白互作网络分析了四种ARSA过表达细胞模型(c.925G,c.925G> A, c.925G> T, c.925G> C)的转录组，结果表明:c.925G处的突变引起与能量代谢，离子结合，囊泡转运和转运相关的分子变化。最后，整合研究一与研究二的研究方法，我们提出了一套鉴定神经系统单基因病致病位点及分析其功能的策略，该策略结合了细胞生物学与生物信息学的方法，能够更加全面地揭示突变影响的生物学功能以及疾病的发病机制。
Monogenetic disease refers to a genetic disease controlled by a pair of alleles. The mode of inheritance conforms to Mendel's law of inheritance, so it is also known as Mendel's disease, showing obvious familial transmission. In the big category of single gene disease, the single gene disease involving the nervous system is called the single gene disease of the nervous system. The clinical needs and scientific research status of single-gene diseases of the nervous system are very important. So far, more than 6,000 single-gene genetic diseases have been identified, of which more than half are related to the nervous and mental systems. Single gene of nervous system diseases and more rare, highly genetic heterogeneity and clinical heterogeneity, the etiology and pathogenesis of many diseases has not been clarified, and has the characteristics of familial and permanent, death, disability, birth defects and to fool rates are very high, treatment difficulty, harm, posed great challenges to the clinical diagnosis and treatment. In this background, this study attempts to identify the pathogenic sites of a clinical highly suspected case of single gene of nervous system diseases by using whole exon sequencing technology, and to explore the biological functions of the sites, and a set of research strategies that can be applied to single gene genetic diseases of nervous system are summarized.
This study consists of two parts: 1) identify the pathogenic genes of single-gene diseases of the nervous system by whole exon sequencing. In this study, suspected pathogenic sites were obtained through sample collection, whole exon sequencing, analysis of sequencing data, screening of pathogenic candidate mutations, genetic verification and other processes.2) biological function verification of pathogenic mutations. In this stu街，one gene overexpression vector and three mutated vectors were constructed and transfected into the HEK293 cell line by transient transfection. The transcriptome data were analyzed by RNA-seq technology. According to the gene expression amount, WGCNA was used to carry out the weighted gene co-expression network analysis to obtain the gene co-expression modules of different groups, that is,some genes with similar expression changes and related functions. Finally, the gene modules of interest were annotated with biological functions.
The results of this study are as follows (1) in this study through in A family with autosomal recessive inheritance patterns, the method of using the fully sequenced exons risk loci identified in the proband of suspected ARSA NM_ 000487. 5: c. 925 g>A p. E 309 k, the site is located in disease and metachromatic leukodystrophy (MLD) ARSA genetically related, through literature review, found that in the previous study, ARSA c. 925 g (c. 925 g>A, c. 925 g>T,c.925G> c) has been reported to cause late infancy MLD. 2) based on the current lack of sufficient biological function studies on this pathogenic site, this study constructed cell models by mutation, and analyzed the transcriptomes of four ARSA-overexpressed cell models (c.925G,c.925G> A,c.925G> T,c.925G> c) through RNA-seq, WGCNA, Hub genes and PPI networks. c. 925 g mutation and energy metabolism, ion, vesicle transport: and related molecular changes. Finally, by integrating the research methods of study 1 and Study 2, we propose a complete set of strategies for identifying pathogenic mutations and functional analysis, which combine cell biology experiments with bioinformatics methods to reveal more comprehensively the biological function of mutations' effects and the pathogenesis of diseases.
The identification and functional study of the pathogenic loci of single-gene hereditary diseases in the nervous system can effectively identify the pathogenic loci of single-gene hereditary diseases and reveal their biological functions, providing clues for the study of the molecular mechanism of diseases.
|Keyword||单基因遗传病 全外显子组测序 功能分析 致病突变鉴定|
|Place of Conferral||中国科学院心理研究所|
|张译丹. 神经系统单基因病致病突变的鉴定与功能分析—— 以异染性脑白质营养不良疾病为例[D]. 中国科学院心理研究所. 中国科学院心理研究所,2020.|
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