The c.119-123dup5bp mutation in human gamma-C-crystallin destabilizes the protein and activates the unfolded protein response to cause highly variable cataracts

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 CRYGC..., 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 ..., , # 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 pulveru...