| ?Objective?With the rapid development of next generation sequencing technology,a large amount of genetic data has been generated and has not been well utilized.In the early stage of the experiment,next generation targeted sequencing of patients with intracerebral hemorrhage was performed.Most of the targeted genes were genes with unknown association with intracerebral hemorrhage.How to find out the pathogenic genes and mutations of intracerebral hemorrhage from thousands of variants is a difficult problem for researchers.The purpose of this study is to use a variety of bioinformatics technologies to comprehensively analyze the data generated by next generation sequencing,thereby selecting genes that can be prioritized for further experiments,and providing an alternative process for gene prioritization.?Methods and results?The subject of this experiment was all the called variants yieled from the next generation sequencing of 394 patients with cerebral hemorrhage and 223 normal controls.We focused mainly on exonic variants.In this study,Phenolyzer was used to perform literature mining.GO and pathway enrichment was also performed.At the same time,genes that specifically changed in cerebral hemorrhage were obtained through analysis of GEO data.Finally,a statistical method based on gene-based burden test further confirmed the results of the above data analysis.This study found that genes related to intracerebral hemorrhage were focused on COL3A1,COL4A6,COL4A4,COL4A3,COL1A2,COL4A5,MMP2,MMP9,COL2A1,COL1A1,COL6A1,etc.in the literature? In GO and pathways analysis the type 4 collagen gene,COL3A1,COL1A2 and COL24A1 belong to the same group of COL4A1 and COL4A2.Statistically significant differences in cases and controls were found in the COL1A2,COL4A5,and COL5A3 genes in SKAT.When ExAC data were used as controls,COL1A1 and COL3A1 were significantly different in all population and East Asian populations,while COL4A5 was only found to be significant in East Asian populations.?Conclusion?Our results show that the comprehensive use of various means of bioinformatics can effectively narrow the range of genes and variants for sequential study.However,the evidence of bioinformatics is limited.To prove the pathogenecity of genes more work is required with functional experiments and genetic evidence.COL1A1,COL3A1,COL1A2,COL4A5,COL5A3,MMP1 and MMP9 were prioritized as the genes for future functional experinments.?Objectives? CRISPR/Cas9 is a gene editing technology that has emerged in recent years.Using CRISPR/Cas9 researchers can efficiently and quickly build a variety of models.Zebrafish gradually emerges as a new model animal for the study of cardiovascular development.At present,there are not many known genes for intracerebral hemorrhage.Based on our previous experiment results,we suggest that COL1A2 may be a causal gene for intracerebral hemorrhage.Therefore,the purpose of this experiment is to construct a zebrafish model using CRISPR/Cas9 technology to study the role of COL1A2 gene in intracerebral hemorrhage.?Methods and results? By injecting a certain dose of sg RNA and Cas9 m RNA into a single cell zebrafish embryo by microinjection,the phenotypes of skeletal dysplasia,cardiac edema,and hemorrhage were observed during embryonic development.At the same time there is no such phenotype in the control zebrafish.The zebrafish of the COL4A1 knockout using CRISPR/Cas9 system showed also vascular malformation and hemorrhage.This result indicates that COL1A2 may be involved in the vascular development of zebrafish,which is similar to that of COL4A1.At the same time,plasmids were constructed and transfected in 293 T cells,we found that they do not affect the expression of genes,indicating that COL1A2 mutation may affect the function of blood vessels through other mechanism,which needs further study.?Conclusion? Our results suggest that COL1A2 may be involved in the development of blood vessels,and the specific mechanisms that COL1A2 mutation affect vascular function causing bleeding needs further research. |
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