Whole-exome Sequencing Identifiy Novel Mutations In Caustive Gene EYS For RP And Function Study Of Causitive Gene For PCG

Genetic disease, which is caused by changes in genetic material, can transfer from parental to offspring. So far, there are more and more eye diseases associated with genetic, such as the retinoblastoma, retinitis pigmentosa(RP, OMIM226800), primary congenital glaucoma(PCG, OMIM231300), congenital red-green color blindness, high myopia. Among them, RP and PCG are the very serious genetic blinding disease. Lots of RP and PCG causative genes have been identified, but these genes cannot fully explain all of the patient’s illness, which indicates that the novel causative genes or mutations are not found.The search for causative genes of disease is always by positional cloning and functional cloning, which can give clues for the molecular pathogenesis of disease. But the traditional methods of causative genes have certain limitations, such as small family, unclear physiological and biochemical mechanism of disease, and so on. With the development of sequencing technology, whole genome technology exons(WES) increasingly appeared in the study of genetic diseases. WES is a method which can capture the exon region of whole exome and then for high-throughput DNA sequencing. In addition, the technology also has the advantages of high sensitivity, low cost and can found that the known and unknown causative genes.This research intends to use WES technology in patients with RP and PCG sequencing, in order to find novel causative genes s or mutations, and further to study the function of causative genes by cell biology and animal models. All of the results are as following:1) In the India RP patients, we found that novel mutations of the known causative gene EYS, which may lead to the occurrence of the RP. These mutations included three pairs of compound heterozygous mutations c. [8422G>A];[7868G>A], c.[4606C>G];[5038A>G] and c [1418G>T];[297CC>T], 7 homozygous mutations c. [1872G>A], c.[8455del A], c.[9059T>C], c.[8388C>A], c.[7187G>C], c.[2259+1 G>A] and c.[3024C >A].2) In order to study the function of novel causative gene P-X, we did cell biology experiments and in vivo experiments. Immunofluorescence results showed that the gene is mainly expressed in cytoplasm and expressed strongly in the retinal photoreceptor cells and ganglion cells of human; and the same in the retina, corner, iris of mouse. By construct the mutant plasmids, we successfully verified that the mutations changed the protein expression. The mutation c.[58C>T] made protein expression lost, and c.[232A>G] showed abnormal distribution in the cells.3) For in vive gene function research, we used morpholino(MO) method in the zebrafish to knockdown the P-X expression. The results showed that the development of zebrafish eyes are affected, characterized by smaller eyes, ganglion cells, rod cells and cone cells decreased, abnormal tissue structure. In order to fiend the reason, we detected the cell apoptosis and proliferation by the Tunel test and phosphorylation protein antibody. The results showed that P-X suppressed the retinal cell proliferation. These implied that P-X may be involved in the pathogenesis of PCG by affected retinal cells proliferation. But the mechanism still needs further research.From what has been discussed above, we give the main conclusions as following:1) By WES, we found the novel mutations including 3 compound heterozygous mutations and 7 homozygous mutations of known causative gene EYS in India RP patients, which expanded the mutation spectrum of RP causative gene;2)The mutations c.[58C>T] and c.[232 A>G] in likely causative gene P-X for PCG may be involved in the development of PCG; and by the cell biology experiments and zebrafish model, we speculated that P-X may play an important role in the cell growth and differentiation of eye tissue.