Abstract
Purpose :
Childhood lamellar cataracts are the most prevalent cataracts. These cataracts are associated with mutations in the heat shock transcription factor 4 (Hsf4). We have generated lamellar cataract mouse model by introducing R116H mutation. The cataract phenotype is subtle, with opacities confined to few lamellae in the lens nucleus. In this study, we investigate the transcriptome of the developing mutant lens to understand molecular progression that leads to cataractogenesis.
Methods :
The lens development in the transgenic mutant (R116H) was followed by 2D-gel analysis of single lens proteins. We used SMARTer stranded total RNA-seq strategies (Takara Bio USA Inc) for transcriptomic analysis of developing ocular lens at post-natal day2, (D02), day10 (D10) and day25 (D25) stages. The data was processed (FastQC) and annotated (Ensembl v84) and analysed using edgeR(TMM).
Results :
Comparison of protein coding transcripts in the WT and the mutant lens, does not show any significant quantitative changes at D02 (20137-WT and 21751- R116H), D10 (21369-WT and 21543-R116H) or at D25 (21509-WT and 21724-R116H). However, we see alterations in the total expression patterns of transcripts unique to each temporal stage. For example, The number of unique transcripts in WT at D02, D10 and D25 are 79, 128 and 195 respectively. In the mutant, they are 44, 20 and 67 suggesting an inhibited program of differentiation. Unsupervised clustering indicates that crystallin gene expression increase appreciably from D02 to D10 and then dwindles by D25 in WT. In the mutant, the crystallin expression is poor both at D02 and D10, yet at D25 we see an increase in selective crystallin transcripts. This inference is also reinforced by the examination of the 2D gel patterns of these predominant proteins. Interestingly, transcript levels of Filensin (Bfsp1) (associated with early onset nuclear cataracts) and DNase 2B, that is associated with nuclear degeneration in terminal differentiation were decreased.
Conclusions :
Based on the complete analysis of the gene expression profiles of the protein coding transcripts at three developmental stages in the WT and the mutant lens we see stalled developmental programs associated with cataractogenesis in the Lamellar cataract transgenic mouse paradigm.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.