研究目的
To identify and confirm CRB2 as a novel causative gene for retinitis pigmentosa (RP) and analyze its pathogenic effects, including how the CRB2 p.R1249G mutation leads to RP through mechanisms like epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells.
研究成果
The CRB2 p.R1249G mutation causes retinitis pigmentosa by disrupting CRB2 protein stability, inducing epithelial-mesenchymal transition in RPE cells, leading to RPE dysfunction, impaired phagocytosis, increased apoptosis, and ultimately retinal degeneration. This establishes CRB2 as a novel gene associated with non-syndromic RP and provides insights into the role of EMT in RP pathogenesis.
研究不足
The in vitro cellular model may not fully replicate in vivo conditions, and the study did not include in vivo animal models or identification of CRB2 mutations in other RP families to further support pathogenicity. The mechanism of how CRB2 mutation affects photoreceptors and Müller cells was not investigated due to time constraints.
1：Experimental Design and Method Selection:
The study used whole exome sequencing (WES) to identify mutations in a consanguineous family with RP, followed by in vitro cellular studies to investigate the functional effects of the identified CRB2 mutation. Methods included genetic analysis, cell culture, transfection assays, RT-PCR, immunoblotting, immunofluorescence, phagocytosis measurement, apoptosis assays, cell cycle analysis, and monitoring of cell proliferation and migration.
2：Sample Selection and Data Sources:
Two patients and three unaffected participants from a Chinese consanguineous family (family LXH) were included. Peripheral venous blood samples were collected for DNA extraction. An additional 423 unrelated Chinese controls were recruited for prevalence testing.
3：List of Experimental Equipment and Materials:
Equipment included HiSeq 2000 platform for WES, confocal microscopes (LSM 510 and Olympus IX70), flow cytometers (gallios flow cytometry), real-time PCR system (StepOne Plus), and xCELLigence system for cell monitoring. Materials included QIAmp DNA Mini Blood Kit, SureSelect Human All Exon 50 Mb Kit, Lipofectamine? 2000, TRIzol reagent, various antibodies, and cell culture reagents.
4：Experimental Procedures and Operational Workflow:
DNA was extracted from blood samples, WES was performed on the proband, bioinformatics analysis filtered variants, and mutation validation was done via Sanger sequencing. In vitro studies involved transfecting ARPE-19 cells with mutant and wild-type CRB2 plasmids, followed by assays for mRNA and protein expression, phagocytosis, apoptosis, cell cycle, proliferation, migration, and EMT biomarkers after TGF-β1 treatment.
5：Data Analysis Methods:
Statistical analysis used Student's T-test with GraphPad Prism software. Protein expression was quantified with Image J software. Bioinformatics tools included SIFT and PolyPhen-2 for mutation prediction, and sequence alignment with Vector NTI Advance?.
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SureSelect Human All Exon 50 Mb Kit
50 Mb
Agilent Technologies
Used for whole exome sequencing to capture and sequence exonic regions.
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StepOne Plus Real-time PCR System
Plus
Applied Biosystems
System for performing real-time PCR to quantify gene expression.
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LSM 510 confocal microscope
510
Carl Zeiss
Used for immunofluorescent staining and image collection.
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Olympus IX70 confocal laser-scanning microscope
IX70
Olympus
Used for imaging in phagocytosis assays.
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HiSeq 2000
2000
Illumina
Platform for high-throughput sequencing in whole exome sequencing.
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QIAmp DNA Mini Blood Kit
Mini
Qiagen
Used for genomic DNA extraction from blood samples.
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Lipofectamine? 2000
2000
Invitrogen
Transfection reagent for introducing plasmids into cells.
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TRIzol reagent
Invitrogen
Used for RNA isolation from cells.
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gallios flow cytometry
gallios
Beckman Coulter
Used for apoptosis assays, cell cycle analysis, and measurement of mitochondrial transmembrane potential.
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xCELLigence system E-Plate
E-Plate
Roche
Used for real-time monitoring of cell proliferation and migration.
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GraphPad Prism
version 4.0
GraphPad Software
Software for statistical analysis of experimental data.
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Image J software
Software for quantifying protein expression from immunoblotting.
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