2019 Fiscal Year Research-status Report
Novel mitochondrial ribosomal genes are associated with mitochondrial disorders
| Project/Area Number |
19K23954
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| Research Institution | Juntendo University |
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Principal Investigator |
ヌルン・ナハール ボルナ 順天堂大学, 医学(系)研究科(研究院), 博士研究員 (30849609)
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| Project Period (FY) |
2019-08-30 – 2021-03-31
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| Keywords | Mitochondrial Disorders |
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| Outline of Annual Research Achievements |
In this research project, we have done WGS, WES, and RNA sequencing to identify the candidate genes of mitoribosomal subunits. I performed Sanger sequencing to validate the variants in patients and the respective family members, and haplotype phasing in two patients due to unavailable DNA from family members. I analyzed the expression of the genes by qRT-PCR. SDS-PAGE and BN-PAGE immunoblotting were done to observe the protein level and assembly of the OxPhos complexes. I analyzed the profile of mitoribosomal subunits by sucrose density gradient analysis. I did luciferase assay to measure ATP under glucose, or galactose media. Oxygen consumption rate by a flux analyzer was done in patients’ fibroblasts and lentiviral-transfected cells. I also performed the stable expression of wild-type mitoribosomal genes in patients’ fibroblasts by lentiviral-mediated transduction. Protein quantitation was done by mass spectrometry.
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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
I have been doing experiments and analyzing data as described in the proposal and acquired expected results. I am hopeful of completing this project on time.
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| Strategy for Future Research Activity |
I will complete the analysis of whole-genome sequencing, proteomic data, and the effect of the variants on protein structure using protein structural analyzing software with the available protein structure on protein data bank (PDB). I will check the reproducibility of the experiments and start the study to generate CRISPR/Cas9 KO cell lines of mitochondrial ribosomal genes. I have plan to use the CRISPR/Cas9 KO cell lines, and perform detailed transcriptome-wide (e.g., DNA microarray or RNA-seq) and functional analyses (e.g., immunoblotting, sucrose density gradient analysis, pull-down). I also have plan to manipulate zebrafish targeted mitochondrial ribosomal genes using CRISPR/cas9-based genome editing technology and investigate the phenotypic correlation with disease pathogenesis.
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| Causes of Carryover |
Reason: Due to the COVID-19 pandemic, EUROMIT-2020 was postponed. I couldn’t attend the conference. So far, my experiments were going well and did not cost much for optimization and acquire data.
Plan: In the future, I will utilize this grant for CRISPR/Cas9 Knockout (KO) and zebrafish KO study optimization, data collection, and analysis.
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