Identification And Functional Study Of A Pathogenic Gene Associated With High Myopia In Uyghurs Based On Epidemiological Survey And Whole-exome Sequencing

Objective: High myopia(HM)is a serious blinding eye disease.In recent years,the onset age of myopia and high myopia is earlier and the prevalence is significantly increased.Genetic factors play an important role in the occurrence and development of HM.This study aimed to investigate the prevalence and risk factors for myopia and high myopia among Han and Uyghur students in Xinjiang,China.In addition,Whole exome-sequencing(WES)was used to identify a candidate gene for a large family of Uyghur with non-syndromic HM.And the functions of candidate pathogenic genes were verified by in vitro and in vivo experiments.In order to fill the gap of myopia epidemiological data in Xinjiang,enrich the high myopia gene database,and provide basis for the diagnosis and treatment of high myopia.Methods: Part 1:(1)This cross-sectional study with a multistage,stratified cluster sampling method was completed in Xinjiang,China.Visual acuity and noncycloplegic refraction were measured.In total,84,033 participants aged 4-23 years were included in the final analysis.The crude and sex-and age-adjusted prevalence of myopia and high myopia in Han and Uyghur students were compared.(2)Logistic regression analyses were applied to identify risk factors associated with myopia and high myopia.Part 2:(1)Starting from probands of early-onset HM in Uyghur children identified by an epidemiological survey,an Uyghur HM family containing 19 members(including 9 cases of HM)in 4 generations was found.A complete ophthalmological examination was performed to obtain clinical information,and the family genogram was plotted.(2)WES was performed on 4 HM cases and 2 controls among the core family members.The relevant genes responsible for high myopia were screened in combination with bioinformatic analysis.(3)Familial co-segregation of candidate genes among all family members was conducted using Sanger sequencing,and the accuracy of second-generation sequencing was verified.(4)Multiplex PCR targeted amplicon sequencing(MTA-seq)was performed among 100 sporadic HM cases and 100 normal controls,so as to verify the presence of the pathogenic genes and mutant loci in sporadic HM cases and normal controls as identified in the HM family,and to search for other mutant loci of the pathogenic gene.(5)The identified mutations and loci were subjected to pathogenicity prediction,conservativeness analysis,mutant loci distribution analysis,and mutant protein simulation and modeling,in order to speculate the effects of gene mutation on the structure and function of the protein encoded by the gene.Part 3:(1)Real-time quantitative PCR(q RT-PCR),western blot(WB),immunofluorescence(IF),and immunohistochemistry(IHC)assays were performed to verify the expression of gene Ppef2(protein phosphatase with EF-hand domain 2)in various tissues of the mouse eye.(2)PPEF2 wild-type,point mutant lentivirus with pathogenic mutant loci was constructed to transiently transfect Human Embryonic Kidney 293 T cells(293T)and to stably transfect Adult Retinal Pigment Epithelial cell line-19(ARPE-19)cells,respectively,so as to construct cell lines stably expressing the target gene.(3)q RT-PCR,WB,and IF assays were performed to verify the effects of PPEF2 mutation on m RNA and protein expressions as well as subcellular localization.(4)The effects of mutation on cell biological behaviors were verified by scratch,CCK-8,plate cloning,and flow cytometry assays of apoptosis and cell cycle.(5)An FDM mouse model was built,and the diopter and ocular biological parameters were measured in vivo to confirm the success of the model.q RT-PCR,WB,IF,and IHC assays were used to preliminarily verify the association between gene Ppef2 in vivo and myopic phenotype.Results: Part 1:(1)The overall age-and sex-adjusted prevalence of myopia and high myopia were 47.70%(95%CI: 47.67-47.74)and 2.55%(95%CI: 2.54-2.56),respectively.(2)The crude prevalence of myopia in the Han population was more than twice that of the Uyghur population(57.01% vs 26.88%,P<0.0001).The crude prevalence of high myopia in the Han population was nearly four times that of the Uyghur population(2.85% vs 0.72%,P<0.0001).(3)The mean SE decreased in both the Han(P<0.001)and the Uyghur populations(P<0.001)as age increased,and the prevalence of myopia and high myopia increased as age increased.(4)Han ethnicity,age,female sex,higher education level and living in urban areas were found to be positively associated with myopia and high myopia.Living in northern Xinjiang was found to be positively associated with myopia but negatively associated with high myopia.Part 2:(1)WES was used to identify a rare heterozygous missense mutation,c.875A>G(p.H292R),in gene PPEF2,which was consistent with family co-segregation in the Uyghur HM family.Moreover,the mutation was absent in the 100 normal controls.(2)MTA-seq identified another rare heterozygous missense mutation,c.1959C>G(p.N653K),in gene PPEF2,in the 100 sporadic HM patients,which was absent in the 100 normal controls.(3)Both mutations were located in important functional and structural domains of protein PPEF2,highly conserved,with pathogenicity prediction as harmful.Protein simulation and modeling showed that both underwent varying degrees of protein structural alterations.Part 3:(1)Gene Ppef2 was expressed in the retinal pigment epithelium and choroid and the retina,but not in the cornea,iris,crystal,and sclera of mice.IF and IHC assays showed that protein PPEF2 was expressed in the optic ganglion cell layer,inner reticular layer,inner nuclear layer,outer reticular layer,outer nuclear layer,the inner segment of optic rod cells,and retinal pigment epithelium.Among them,it was strongly expressed in the inner segment layer formed by photoreceptor protrusions,as well as in the outer nuclear layer.(2)In both293 T and ARPE-19 cells,the A875 G mutation in gene PPEF2 resulted in reduced expression levels of m RNA and protein but had no effect on protein subcellular localization.(3)The A875 G mutation in gene PPEF2 resulted in decreased migratory capacity,proliferative capacity,and promote apoptosis in ARPE-19 cells.(4)Down-regulated expressions of PPEF2 protein and m RNA were observed in the FDM mouse model.Conclusion:(1)Compared to the Uyghur population,the Han population living in the same area had a higher prevalence of myopia and high myopia.The prevalence of myopia among the Uyghur population showed a more remarkable increasing trend than that among the Han population in Xinjiang.(2)Han ethnicity,age,female sex,higher education level and living in urban areas were found to be positively associated with myopia and high myopia.Living in northern Xinjiang was found to be positively associated with myopia but negatively associated with high myopia with myopia and high myopia.(3)Variants in PPEF2 cause non-syndromic HM in Uyghurs.(4)The variant in PPEF2 might cause HM through regulating the migratory,proliferative and apoptosis of myopia-related cells.