The c.119-123dup5bp mutation in human gamma-C-crystallin destabilizes the protein and activates the unfolded protein response to cause highly variable cataracts
Data files
Aug 23, 2024 version files 33.29 GB
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MC37_USR18004116L_HGVVMDMXX_L1_1.fq.gz
897.68 MB
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MC37_USR18004116L_HGVVMDMXX_L1_2.fq.gz
928.47 MB
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MC37_USR18004116L_HGVVMDMXX_L2_1.fq.gz
911.05 MB
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MC37_USR18004116L_HGVVMDMXX_L2_2.fq.gz
938.61 MB
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MC39_USR18004117L_HGVVMDMXX_L1_1.fq.gz
966.22 MB
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MC39_USR18004117L_HGVVMDMXX_L1_2.fq.gz
987.58 MB
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MC39_USR18004117L_HGVVMDMXX_L2_1.fq.gz
979.88 MB
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MC39_USR18004117L_HGVVMDMXX_L2_2.fq.gz
997.76 MB
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MC42_USR18004118L_HGVVMDMXX_L1_1.fq.gz
899.76 MB
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MC42_USR18004118L_HGVVMDMXX_L1_2.fq.gz
945.74 MB
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MC42_USR18004118L_HGVVMDMXX_L2_1.fq.gz
915.66 MB
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MC42_USR18004118L_HGVVMDMXX_L2_2.fq.gz
959.98 MB
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MS55_USR18004119L_HGVVMDMXX_L1_1.fq.gz
902.74 MB
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MS55_USR18004119L_HGVVMDMXX_L1_2.fq.gz
945.27 MB
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MS55_USR18004119L_HGVVMDMXX_L2_1.fq.gz
916.47 MB
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MS55_USR18004119L_HGVVMDMXX_L2_2.fq.gz
956.07 MB
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MS56_USR18004120L_HGVVMDMXX_L1_1.fq.gz
833.39 MB
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MS56_USR18004120L_HGVVMDMXX_L1_2.fq.gz
866.96 MB
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MS56_USR18004120L_HGVVMDMXX_L2_1.fq.gz
844.25 MB
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MS56_USR18004120L_HGVVMDMXX_L2_2.fq.gz
875.59 MB
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MS58_USR18004121L_HGVVMDMXX_L1_1.fq.gz
971.04 MB
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MS58_USR18004121L_HGVVMDMXX_L1_2.fq.gz
1.01 GB
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MS58_USR18004121L_HGVVMDMXX_L2_1.fq.gz
988.75 MB
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MS58_USR18004121L_HGVVMDMXX_L2_2.fq.gz
1.02 GB
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qRT_PCR_Data.zip
19.94 MB
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README.md
11.32 KB
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WC33_USR18004113L_HGVVMDMXX_L1_1.fq.gz
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WC33_USR18004113L_HGVVMDMXX_L1_2.fq.gz
855.26 MB
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WC33_USR18004113L_HGVVMDMXX_L2_1.fq.gz
834.35 MB
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WC33_USR18004113L_HGVVMDMXX_L2_2.fq.gz
857.48 MB
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WC65_USR18004114L_HGVVMDMXX_L1_1.fq.gz
870.05 MB
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WC65_USR18004114L_HGVVMDMXX_L1_2.fq.gz
899.40 MB
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WC65_USR18004114L_HGVVMDMXX_L2_1.fq.gz
880.99 MB
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WC65_USR18004114L_HGVVMDMXX_L2_2.fq.gz
907.36 MB
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WC67_USR18004115L_HGVVMDMXX_L1_1.fq.gz
939.02 MB
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WC67_USR18004115L_HGVVMDMXX_L1_2.fq.gz
984.07 MB
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WC67_USR18004115L_HGVVMDMXX_L2_1.fq.gz
953.81 MB
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WC67_USR18004115L_HGVVMDMXX_L2_2.fq.gz
996.42 MB
Abstract
Ordered cellular architecture and high concentrations of stable crystallins are required for the lens to maintain transparency. Here we investigate the molecular mechanism of cataractogenesis of the CRYGC c.119-123dupGCGGC (p.Cys42AlafsX63) (CRYGC5bpd) mutation. Lenses were extracted from wild-type and transgenic mice carrying the CRYGC5bpdup minigene and RNA was isolated and converted into cDNA. Expression of genes in the unfolded protein response (UPR) pathways was estimated by qRT-PCR and RNA seq and pathway analysis was carried out using the Qiagen IPA website. P3W Transgenic mice exhibited phenotypic diversity with a dimorphic population of severe and clear lenses. PCA of RNA seq data showed separate clustering of wild-type, clear CRYGC5bpd, and severe CRYGC5bpd lenses. Transgenic mice showed differential upregulation in Master regulator Grp78 (Hspa5) and downstream targets in the PERK-dependent UPR pathway including Atf4 and Chop (Ddit3), but not GADD34. Thus, high levels of CRYGC5bpd transgene expression in severely affected lenses induce UPRer and UPRmt stress responses primarily through the PERK-dependent and Atf4/Atf5/Ddit3 pathways respectively, inducing autophagy and apoptosis and thence congenital nuclear cataracts. This effect is correlated to CRYGC5bpd transgene expression, offering insight into cataract pathogenic pathways and variation in cataract severity in humans.
README: The c.119-123dup5bp mutation in human gamma-C-crystallin destabilizes the protein and activates the unfolded protein response to cause highly variable cataracts
https://doi.org/10.5061/dryad.rn8pk0pmm
Description of the data and file structure
The RNASeq and qRT-PCR files included refer to mice transgenic for a CRYGC c.119-123dupGCGGC (p.Cys42AlafsX63) (CRYGC5bpd) mutation. Lenses were extracted from wild-type and transgenic mice carrying the CRYGC5bpdup minigene and RNA was isolated and converted into cDNA and submitted to Novogene for RNASeq analysis. The descriptions of the mice are given in Ma, Z. et al. Overexpression of human γC-crystallin 5bp duplication Disrupts Lens Morphology in Transgenic Mice. Invest Ophthalmol Vis Sci 52, 5269-5375 (2011).
Identification of CRYGC as the causative gene is described in Ren, Z. et al. A 5-base insertion in the γC-crystallin gene is associated with autosomal dominant variable zonular pulverulent cataract. Hum Genet 106, 531-537 (2000). The human cataracts are described in Scott, M. H. et al. Autosomal dominant congenital cataract: Interocular phenotypic heterogeneity. Ophthalmology 101, 866-871 (1994).
The files are of two types:
- compressed fastq files and can be read on any software package that processes next-generation sequencing data such as Golden Helix SVS, or sequence alignment tool such as found at the online EMBL Job Dispatcher (https://www.ebi.ac.uk/jdispatcher/).
- compressed qRT-PCR output files are of two types. The raw files are from an Applied Biosystems Vii7 real-time PCR analyzer and are .eds files. The exported files are in MS Excel format (.xls).
The contents of the sequencing files are as follows:
Result contents -【 type】- description
- MC37_USR18004116L_HGVVMDMXX_L1_1.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks transgenic mouse ID:507
- MC37_USR18004116L_HGVVMDMXX_L1_2.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks transgenic mouse ID:507
- MC37_USR18004116L_HGVVMDMXX_L2_1.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks transgenic mouse ID:507
- MC37_USR18004116L_HGVVMDMXX_L2_2.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks transgenic mouse ID:507
- MC39_USR18004117L_HGVVMDMXX_L1_1.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks transgenic mouse ID:500
- MC39_USR18004117L_HGVVMDMXX_L1_2.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks transgenic mouse ID:500
- MC39_USR18004117L_HGVVMDMXX_L2_1.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks transgenic mouse ID:500
- MC39_USR18004117L_HGVVMDMXX_L2_2.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks transgenic mouse ID:500
- MC42_USR18004118L_HGVVMDMXX_L1_1.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks transgenic mouse ID:522
- MC42_USR18004118L_HGVVMDMXX_L1_2.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks transgenic mouse ID:522
- MC42_USR18004118L_HGVVMDMXX_L2_1.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks transgenic mouse ID:522
- MC42_USR18004118L_HGVVMDMXX_L2_2.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks transgenic mouse ID:522
- MS55_USR18004119L_HGVVMDMXX_L1_1.fq.gz 【Read for paired-end sequencing】 gzip file severe cataract lens from 3 weeks transgenic mouse ID:505
- MS55_USR18004119L_HGVVMDMXX_L1_2.fq.gz 【Read for paired-end sequencing】 gzip file severe cataract lens from 3 weeks transgenic mouse ID:505
- MS55_USR18004119L_HGVVMDMXX_L2_1.fq.gz 【Read for paired-end sequencing】 gzip file severe cataract lens from 3 weeks transgenic mouse ID:505
- MS55_USR18004119L_HGVVMDMXX_L2_2.fq.gz 【Read for paired-end sequencing】 gzip file severe cataract lens from 3 weeks transgenic mouse ID:505
- MS56_USR18004120L_HGVVMDMXX_L1_1.fq.gz 【Read for paired-end sequencing】 gzip file severe cataract lens from 3 weeks transgenic mouse ID:506
- MS56_USR18004120L_HGVVMDMXX_L1_2.fq.gz 【Read for paired-end sequencing】 gzip file severe cataract lens from 3 weeks transgenic mouse ID:506
- MS56_USR18004120L_HGVVMDMXX_L2_1.fq.gz 【Read for paired-end sequencing】 gzip file severe cataract lens from 3 weeks transgenic mouse ID:506
- MS56_USR18004120L_HGVVMDMXX_L2_2.fq.gz 【Read for paired-end sequencing】 gzip file severe cataract lens from 3 weeks transgenic mouse ID:506
- MS58_USR18004121L_HGVVMDMXX_L1_1.fq.gz 【Read for paired-end sequencing】 gzip file severe cataract lens from 3 weeks transgenic mouse ID:508
- MS58_USR18004121L_HGVVMDMXX_L1_2.fq.gz 【Read for paired-end sequencing】 gzip file severe cataract lens from 3 weeks transgenic mouse ID:508
- MS58_USR18004121L_HGVVMDMXX_L2_1.fq.gz 【Read for paired-end sequencing】 gzip file severe cataract lens from 3 weeks transgenic mouse ID:508
- MS58_USR18004121L_HGVVMDMXX_L2_2.fq.gz 【Read for paired-end sequencing】 gzip file severe cataract lens from 3 weeks transgenic mouse ID:508
- WC33_USR18004113L_HGVVMDMXX_L1_1.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks wildtype mouse ID:503
- WC33_USR18004113L_HGVVMDMXX_L1_2.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks wildtype mouse ID:503
- WC33_USR18004113L_HGVVMDMXX_L2_1.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks wildtype mouse ID:503
- WC33_USR18004113L_HGVVMDMXX_L2_2.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks wildtype mouse ID:503
- WC65_USR18004114L_HGVVMDMXX_L1_1.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks wildtype mouse ID:535
- WC65_USR18004114L_HGVVMDMXX_L1_2.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks wildtype mouse ID:535
- WC65_USR18004114L_HGVVMDMXX_L2_1.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks wildtype mouse ID:535
- WC65_USR18004114L_HGVVMDMXX_L2_2.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks wildtype mouse ID:535
- WC67_USR18004115L_HGVVMDMXX_L1_1.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks wildtype mouse ID:537
- WC67_USR18004115L_HGVVMDMXX_L1_2.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks wildtype mouse ID:537
- WC67_USR18004115L_HGVVMDMXX_L2_1.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks wildtype mouse ID:537
- WC67_USR18004115L_HGVVMDMXX_L2_2.fq.gz 【Read for paired-end sequencing】 gzip file clear lens from 3 weeks wildtype mouse ID:537
Table-1: For the qRT-PCR the following gives the raw file name and its description.
| File Name | Description |
|---|---|
| UPR_P3W_11072017-1.eds | Raw file |
| UPR_P3W_11072017-1-ViiA7-export | Export file |
| UPR_P3W-3 4 and 6_08032017-1 | Raw file |
| UPR_P3W-3 4 and 6_08032017-1-ViiA7-export | Export file |
Table-2: The following table describes the samples used for qRT-PCR.
| File Name | Sample Name | Description |
|---|---|---|
| UPR_P3W_11072017-1-ViiA7-export | P3W-3-2-WC1 | Wild type mice |
| UPR_P3W_11072017-1-ViiA7-export | P3W-4-6-WC1 | Wild type mice |
| UPR_P3W_11072017-1-ViiA7-export | P3W-6-4-WC1 | Wild type mice |
| UPR_P3W_11072017-1-ViiA7-export | P3W-6-5-WC1 | Wild type mice |
| UPR_P3W_11072017-1-ViiA7-export | P3W-3-1-MC1 | c.119-123dup5p transgenic mice with clear lens |
| UPR_P3W_11072017-1-ViiA7-export | P3W-4-7-MC1 | c.119-123dup5p transgenic mice with clear lens |
| UPR_P3W_11072017-1-ViiA7-export | P3W-6-2-MC1 | c.119-123dup5p transgenic mice with clear lens |
| UPR_P3W_11072017-1-ViiA7-export | P3W-6-2-MC2 | c.119-123dup5p transgenic mice with clear lens |
| UPR_P3W_11072017-1-ViiA7-export | P3W-3-1-MS1 | c.119-123dup5p transgenic mice with severe lens |
| UPR_P3W_11072017-1-ViiA7-export | P3W-4-8-MS1 | c.119-123dup5p transgenic mice with severe lens |
| UPR_P3W_11072017-1-ViiA7-export | P3W-6-3-MS1 | c.119-123dup5p transgenic mice with severe lens |
| UPR_P3W_11072017-1-ViiA7-export | P3W-6-3-MS2 | c.119-123dup5p transgenic mice with severe lens |
| UPR_P3W-3 4 and 6_08032017-1-ViiA7-export | P3W-3-2-C1-WC1 | Wild type mice |
| UPR_P3W-3 4 and 6_08032017-1-ViiA7-export | P3W-4-6-C1-WC1 | Wild type mice |
| UPR_P3W-3 4 and 6_08032017-1-ViiA7-export | P3W-6-4-C1-WC1 | Wild type mice |
| UPR_P3W-3 4 and 6_08032017-1-ViiA7-export | P3W-6-5-C1-WC1 | Wild type mice |
| UPR_P3W-3 4 and 6_08032017-1-ViiA7-export | P3W-3-1-C1-MC1 | c.119-123dup5p transgenic mice with clear lens |
| UPR_P3W-3 4 and 6_08032017-1-ViiA7-export | P3W-4-7-C1-MC1 | c.119-123dup5p transgenic mice with clear lens |
| UPR_P3W-3 4 and 6_08032017-1-ViiA7-export | P3W-6-1-C1-MC1 | c.119-123dup5p transgenic mice with clear lens |
| UPR_P3W-3 4 and 6_08032017-1-ViiA7-export | P3W-6-1-C2-MC2 | c.119-123dup5p transgenic mice with clear lens |
| UPR_P3W-3 4 and 6_08032017-1-ViiA7-export | P3W-3-1-S1-MS1 | c.119-123dup5p transgenic mice with severe lens |
| UPR_P3W-3 4 and 6_08032017-1-ViiA7-export | P3W-4-8-S1-MS1 | c.119-123dup5p transgenic mice with severe lens |
| UPR_P3W-3 4 and 6_08032017-1-ViiA7-export | P3W-6-3-S1-MS1 | c.119-123dup5p transgenic mice with severe lens |
| UPR_P3W-3 4 and 6_08032017-1-ViiA7-export | P3W-3-2-C1-WC1 | c.119-123dup5p transgenic mice with severe lens |
Code/Software
NA
Access information
NA
Methods
RNA isolation, cDNA synthesis, and qRT-PCR
Total RNA was isolated using an RNA isolation kit (The RNeasy Plus Mini Kit; Qiagen, Valencia, CA) and quantified using a spectrophotometer (Nanodrop 2000C; ThermoFisher). A first-strand cDNA was synthesized from approximately 0.5mg of total RNA by cDNA synthesis kit (Super III first-strand synthesis for RT PCR kit; Invitrogen) according to the manufacturer's protocol. qRT-PCR was performed using Applied Biosystems ViiA7 Real-Time PCR system with the following amplification conditions: an initial incubation of the samples at 50°C for 2min and denaturation at 95°C 15min followed by 40 cycles of denaturation, annealing, and extension at 95°C 15sec, 60°C 30sec, and 72°C 30sec. Gapdh was used as an endogenous control for normalizing the target mRNA. The relative expression of each target gene was calculated using the 2^(∆∆Ct) method. The primers were standardized, and efficiencies were tested before performing qRT-PCR.
RNA-Seq
About 200ng of RNA samples were used for RNA seq. analysis. Total RNA samples were purified from the lenses of 3-week-old wild-type and KO mice (n = 3) using a Qiagen kit. After passing quality control criteria, RNA-sequencing was carried out using an Illumina NovaSeq platform utilizing a paired-end 150 bp sequencing strategy (Novogene Corporation Inc., Sacramento, CA, USA). Analysis was performed using Partek Flow (https://partekflow.cit.nih.gov/flow) on the NIH Biowulf supercomputing cluster.