DOI: 10.5061/dryad.rn8pk0pmm
title: The c.119-123dup5bp mutation in human gamma-C-crystallin destabilizes the protein and activates the unfolded protein response to cause highly variable cataracts

description: 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.
description: 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. <em>Gapdh</em> 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.
description: # 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](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: 1. 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/](https://www.ebi.ac.uk/jdispatcher/)). 2. 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