2020 Fiscal Year Research-status Report
モデルマウスを用いた、正常眼圧緑内障家系で同定された新規原因遺伝子Xの機能解析
| Project/Area Number |
20K18366
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| Research Institution | 独立行政法人国立病院機構(東京医療センター臨床研究センター) |
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Principal Investigator |
潘 洋 独立行政法人国立病院機構(東京医療センター臨床研究センター), 分子細胞生物学研究部, 研究員 (20866389)
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| Project Period (FY) |
2020-04-01 – 2023-03-31
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| Keywords | Normal-tension glaucoma / Causative gene / Splicing |
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| Outline of Annual Research Achievements |
1.Identification of the novel causative mutation. To identify novel NTG causative genes, whole-exome sequencing analysis was performed. According to the frequency and functional prediction, one mutation in the novel gene X was identified as the causative mutation.
2. The conserved novel mutation exhibits gain of splicing in vitro. To identify the effect of the novel mutation on mRNA splicing, in vitro splicing assay was performed in HEK 293 cells. As expected, the mutation resulted in completed exon skipping, effectively removing 106 bp including the start codon.
3. The novel mutation cause morphology changes in RGCs by OCT. To gain further insight into the function of mutation, we analyzed GC complex thickness, disc & cup volume and number of GC in knock-in and knockout mice. B-circular and B-horizontal of OCT scan revealed that the thickness of RGC fiber layer around optic nerve head (ONH) significantly reduced and disc & cup volume increased dramatically in both knock-in and knockout mice comparing with controls by 2-month of age
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Following the research plan, we have found that the thickness of RGC fiber layer around optic nerve head (ONH) significantly reduced and disc & cup volume increased dramatically, NTG phenotype, in both knock-in and knockout mice comparing with controls by 2-month of age. Moreover, we confirmed the splicing ability in vitro.
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| Strategy for Future Research Activity |
1. The mutation leads to splicing in iPSCs. We have generated the human iPSCs using the peripheral blood lymphocytes obtained from the patients carrying the novel mutation. The splicing ability will be confirmed in iPSCs by RT-PCR and TA-cloning.
2. Expression and intracellular localization in vitro and vivo.The expression and intracellular localization will be analyzed by WB and immunofluorescence in transfected cells. To assay the endogenous expression, immunofluorescence will be performed in the mouse and monkey paraffin section
3. The novel mutation cause morphology changes in RGCs by OCT. To confirm the morphology changes, we will continue to observe the GC complex thickness, disc & cup volume and number of GC in knock-in and knockout mice by OCT in 4-month old and 6-month old mice.
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| Causes of Carryover |
We planned to start culture iPSCs and confirm the splicing ability in the first year, however, both knock-in and knockout mice first appear the phenotype with litter size. So, we first focus on the mice. Later we will start to culture iPSCs. After isolating RNA, RT-PCR and TA-cloning following sanger sequencing will be performed.
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