Study On Identification Of High Myopia-related Genes And Evaluation Of VIPR2 Function In Myopia

High myopia and its complications already become the primary reason behind irreversible visual impairment and even blindness in my country.Nevertheless,the pathogenic mechanism of high myopia is not clear.The effective prevention and treatment techniques,as well as drugs,are still extremely restricted.Hence,searching for the pathogenic factors of myopia and exploring the molecular mechanism of its pathogenesis is highly important.To search the pathogenesis of high myopia,this dissertation looks for candidate disease genes from genetics and experimentally determines the role of disease genes in myopia.The research outcomes of this dissertation have been summarized as follows:1.To verify and discuss the disease genes of high myopia.We explored the relationship between 17 single nucleotide sites in FGF10,PDGFRA,PARP8,ZC3H11B,SEMA4F and SPTBN1 genes and high myopia.The carriers of rs12517396AA and rs10462070GG showed lower susceptibility to high myopia(P=0.048,OR=0.370;P=0.032,OR=0.346).The rs2114039 of FPDGFRA gene was also significantly associated with high myopia(P=3.02�10-08,OR=0.579);we also confirmed that the 7SNP loci in the ZC3H11B,SEMA4F and SPTBN1 genes were not associated with Han population in the southwestern region of China.2.The role of VIPR2 gene in the formation of myopia.In this disserrtation,through sequencing the entire gene exome of high myopia patients,we screened out multiple pathogenic mutation sites of VIPR2 genes,and the following conclusions were obtained:(1)after the sequencing verification of expanded samples,there were 4 pathogenic mutations(c.G103A:p.E35K,c.G946A:p.G316S,c.G541A:p.D181N,c.A320T:p.D107V)which only presented in patients with high myopia.The conservative analysis of the mutation sites showed that they were all in conservative sequences.(2)In vitro experiments explored the role of VIPR2 in the process of collagen synthesis.The sclera is an important target organ in the formation of myopia.In this dissertation,after knocking down VIPR2 in human-derived primary scleral fibroblast(HFSF)cells,it was found the expression of collagen I reduced.VIPR2mutants(G103A and A320T)also reduced the expression of type I collagen.Mutants can also reduce the expression of PDE7a,SEC61a,VPS33B,and MIA3 related to the synthesis pathway of type I collagen.The genome-wide association analysis(GWAS)results previously reported by our laboratory illustrated that the VIPR2 gene is a crucial disease gene associated with high myopia.We have further studied VIPR2 functions in vivo and in vitro.Firstly,the function of VIPR2 was studied in vivo through a mouse model of common myopia,a gene knockout mouse model,and an environment-induced mouse model,and the following conclusions were obtained:(1)animal model confirmed that Vipr2 is involved in the formation of myopia.A mouse model of form deprivation myopia was constructed.We develop a form deprivation(FDM)mouse myopia model.Through RT-PCR and WB detection,it was found that the VIP protein expression risen,while VIPR2 protein expression reduced.(2)After successfully constructing Vipr2^-/-mice,it was found that the mouse showed signs of nearsightedness(refractive changes,longer eye axis).Long-term follow-up observations found that circadian rhythms of Vipr2^-/-mice still exist in a normal cyclic lighting environment(12h light/12h darkness),but the rhythm cycle is shorter than that of wild-type mice.The emmetropization process of Vipr2^-/-was disrupted,and the diopter was always lower than that of normal mice.(3)The myopia phenotype of Vipr2^-/-male mice is more pronounced in a 24h-dark environment.We found that the circadian rhythm of knockout mice almost completely disappered in that enviroment.Compared with the circulatory lighting environment,the changes of refractive and eye axis were more obvious.The emmetropization process of Vipr2^-/-mice was almost completely destroyed.We further observed the results of scleral tissue in mice.In the circulatory light environment,the scleral results of Vipr2^-/--male mice maintained a normal state,while in a 24h-dark environment,the scleral fibroblasts showed significant changes such as loss of organelles,decreased collagen expression,and sparse collagen bundles.(4)The influence of VIPR2 on proteins related to circadian rhythm regulation.We found that the absence of Vipr2 gene in retinal tissues leads to a decrease in the expression of per2 and per3 genes at the same time.In summary,we have confirmed through animal models and cell lines that VIPR2gene is involved in the formation of myopia.The loss of VIPR2 gene function may disrupt the normal endogenous circadian rhythm of the eye tissues by affecting the circadian rhythm regulation related proteins,and at the same time block the synthesis of scleral collagen,interrupt the process of eye emmetropia,cause abnormal refractive development,and lead to myopia.3.Explore the function of PARP8 in myopia.It was found that rs1195438,rs2404958,rs282544,rs32396,and rs27243 in the PAPR8 gene had strong association with high myopia of the Han population.These five loci are located in the same linkage region.The results of the ACTAA haplotype analysis suggest that the ACTAA carrier had an increasing risk of high myopia(p=2.00�10-04;OR=1.294).The rs27243T>A mutation in the 3'-UTR region of the PARP8 gene generated a new binding site for hsa-mi RNA-410-3p,which regulates PARP8 through negative feedback protein expression.By comparing the detection of high myopia with normal people,it was explored that the expression of PARP8 gene reduced and the expression of hsa-mi RNA-410-3p increased in myopia population.We further explored the signal pathway of PARP8.Through mass spectrometry and CO-IP experiments,we found that PARP8 interacts with ALYREF.ALYREF plays an important role in the process of m RNA transcription and DNA damage repair.At the same time,we also found that the decrease of PARP8 expression will cause changes in cell morphology,decreased expression of Lamin A/C,and changes in the expression of CHK1 and CHK2 proteins related to apoptosis.Therefore,we speculate that the loss of PARP8 function will cause the ALYREF protein-mediated m RNA transcription process to be blocked,resulting in a decrease in cell viability,and then affecting the occurrence and development of myopia.