Characterization Of C21ORF2 In The Pathogenesis Of Retinal Ciliopathy

Hereditary retinal ciliopathy is a group of retinal degeneration disease caused by mutations related to ciliopathy,which lacks effective treatment due to unknown pathogenesis.Recent research suggested C21ORF2 mutation was associated with degeneration in cone and rod cells in retina during infant,and the encoded protein was found to colocalize and to interact with ciliopathy-associated proteins at the photoreceptor transition zone.However,its function and pathogenesis still remained unknown.Our objectives are to investigate the effect of C21ORF2 mutation and knockdown on the expression of proteins and growth of cilia,thus revealing the association between C21ORF2 and retina ciliopathy.During in vitro experiments,two EGFP-labelled plasmids carrying retinal ciliopathy causative mutants（c.218G>C;p.Arg73Pro and c.103del A;p.Ile35Phefs*10）were built and termed p EGFP-C21orf2^G218C and p EGFP-C21orf2^{103del A}.Three C21orf2-specific si RNAs were also generated and transfected into the IMCD3 cells.Compared to the control wild-type plasmid or si RNA,the expression levels of C21ORF2 were reduced in either mutant or si RNA knockdown IMCD3 cilia cells in Western blot,while no expression was observed in GFP-C21ORF2（103del A）.Then,we knocked down C21orf2in IMCD3 cells by si RNA and generated the same cell models with reduced protein expression.The results of immunocytochemistry suggested reduced expression of C21ORF2 can decrease the number and length of cilia,which revealed the vital role of C21ORF2 in cilia formation.During in vivo experiments,we identified the distribution of expression of C21ORF2in mouse tissues,and found C21ORF2 was widely expressed in nearly all mouse tissues,including testis,ovary,lung,kidney,heart,eye,intestinal,brain,skeletal muscles,etc.We determined the expression site of C21ORF2 in retina by immunohistochemistry,which was located in transition zone of photographic cells.To further explore the effect of C21ORF2 mutation on ciliopathy,we generated C21orf2conditional knockout(C21orf2^G218C)and 103del A knock-in(C21orf2^{103del A})mouse models by CRISPR（Clustered Regularly Interspaced Short Palindromic Repeats）/Cas9technique.However,we did not obtain C21orf2^{103del A} mice due to congenital lethality,which suggested 103del A mutation might affect the development of embryo.As for the C21orf2^G218C mice,the results from immunohistostaining and electroretinogram did not show significant morphological degeneration and function loss in retina in mice from one to twelve months.No significant morphological changes and function loss was observed in the twelve months old mice,which suggested C21ORF2（c.218G>C;p.Arg73Pro）mutation may not lead to ciliopathy in mice.The results of our project served as the preliminary evidence for the complicated pathogenesis of ciliopathy,which might provide potential reference to future work.