Identification Of PRSS56 Mutation In A Family With High Myopia And The Study Of The Correlation Between This Gene And Refractive Error

Objectives:The definition of ametropia refers to that when the eye does not use adjustment,the parallel light can not be clearly imaged on the retina after passing through the refractive medium of the eye,but can be imaged in front or behind the retina.It contains hyperopia,myopia and astigmatism.The prevalence of ametropia is increasing rapidly,which exerts a great impact on public health.Among them,high myopia is often accompanied by other serious complications.One third of high myopia patients will have irreversible visual impairment or blindness.There are a lot of genetic ways of ametropia.Genetic research is conducive to gene diagnosis and treatment of ametropia.The present study aims to screen the pathogenic genes in a family with high myopia,identify the pathogenic genes leading to high myopia,and attempt to explore the molecular mechanism of its mutation.Methods:(1)This research collected a family with high myopia,diagnosed as hereditary high myopia after refractive examination,exclusive examination of anterior segment and fundus diseases,and drew a family map.After obtaining the informed consent of each subject,it collected the clinical data and peripheral blood of the patients and their families and extracted DNA.Meanwhile,DNA of three members of a core family was selected for whole-exome sequencing,pathogenic genes were screened,and Sanger validation and co-separation analysis were conducted.(2)Combining previous literature reports,this research sorted out the previous mutation sites of PRSS56,and screened one mutation site that caused myopia(c.G88A(A30T))one that caused hyperopia(c.G926C(W309S)),and the mutation site(c.C827T(A276V))from this high myopia family as the object for further functional verification;(3)The evolutionary conservation and stability of the gene domain and mutation site were analyzed.Furthermore,Pymol software was used to perform homology modeling of the mutant protein and analyze the effect of the mutation site on the protein structure.(4)This research synthesized the full length of PRSS56,constructed a wild-type PRSS56 vector with FLAG on the C-terminal,performed site-specification on the wild-type vector template Mutation,and transiently transfected the wild-type and mutant vectors into HEK-293T cells.(5)The cells were divided into 5 groups,including WT group:wild-type group;c.C827T(A276V)group:high myopia identified in this study,Mutant group;c.G88A(A30T)group:another myopia group;c.G926C(W309S)group:high hyperopia group;NC group:blank control group with only transfection reagent added;(6)This research collected cell culture supernatants 48h after transfection,and measured the concentration of PRSS56 in the supernatant by ELISA to determine the activity of different groups;(7)Four groups of cells except NC were subjected to Western blotting to detect the expression of FLAG and Calnexin and comparison was made;(8)All groups except NC conducted Flag and Calnexin immuno-dual fluorescence detection,confocal microscope was used to observe the cell morphology changes between the groups,and comparison was made;(9)After extracting RNA from cells in each group,this research performed transcriptome sequencing analysis.(10)Using GraphPad Prism 6 for experimental data.The software was used for performing analysis and one-way ANOVA was used for comparison.P<0.05 was considered to have significant and statistical significance.All experiments were independently repeated 3 times.Results:(1)The high myopia family included in the present study has four generations of 27 members,of which 10 patients were diagnosed with high myopia(9 females,1 male),and none of the family members suffered from severe myopia unrelated to high myopia;(2)Three candidate genes were screened out:PRSS56,COL18A1 and HNF1A.It was verified that PRSS56,c.C827T(A276V)and the 7 high myopia patients in the present family achieved family gene co-segregation.(3)Through the analysis of health information,it can be observed that the mutation of PRSS56 c.C827T(A276V)happens to occur in the conserved domain of PRSS56 protein.This site is highly conserved in various species.In the meanwhile,this mutation will destroy the structural stability of the wild-type protein.The 3D conformation of the protein was significantly changed.(4)The concentration of PRSS56 in the cell culture supernatant of each group after 48 hours of transfection:c.G88A(A30T),c.C827T(A276V)and c.G926C(W309S)significantly increased(P<0.01).In addition,the results also showed that c.G926C(W309S)was significantly reduced in comparison with NC group(P<0.01),while WT group was significantly lowered compared with c.G88A(A30T)(P<0.05);(5)The activity of PRSS56 in each group:c.G88A(A30T)and c.C827T(A276V)have roughly the same trajectory,and they all decrease gradually after 1h.The activity of c.G926C(W309S)group decreases gradually,and basically maintains after reaching the minimum at 1h.;(6)According to the results of Western-blot,the flag-labeled PRSS56 fusion protein is only expressed in the membrane fraction structure;c.G88A(A30T),c.C827T(A276V),The PRSS56 fusion protein was highly expressed,which was significantly different from the c.G926C(W309S)group and the WT group(P<0.05);(7)48h after cell transfection,the immunofluorescence-stained cells of each group were displayed on the confocal microscope.There is no difference between Flag and Calnexin,both of which are located in the endoplasmic reticulum.(8)The transcriptome analysis results are as presented follows.The number of differential genes:c.G926C vs c.G88A&c.C827T is 1321,WT vs c.G926C is 157,and WT vs c.C827T is 80.WT vs c.G88A has 49 differential genes.Obviously,c.G926C vs c.G88A&c.C827T is significantly higher than the other three groups.WT vs c.C827T and WT vs c.G88A,among the genes shared by 3 are up-regulated and 6 are down-regulated.Among them,ANKHD1 is strongly associated with PRSS56.The regulatory pathways that each group focuses on are as follows:c.G926C vs c.G88A&c.C827T:Micro Tube cell bone Framework organization;WT vs c.C827T,reaction to calcium ions,precursor metabolites and energy production,WT vs c.G88A,cellular defense response,post-translational protein modification,WT vs c.G926C,oxidative phosphorylation and mitochondria organization.Conclusions:(1)The four generations of autosomal dominant high myopia families recruited in the present study showed clinical phenotypes of the disease,such as diopter≤-6.0D and eye axis length>26mm,and some family members have been associated with high myopia complications such as cataracts,retinal detachment and leopard-like fundus changes,etc.The current work identified a new missense mutation in PRSS5 6 gene for the first time in a Chinese family with high myopia:c.C827T(A276V).This mutation site has a strong association with high myopia.The mutation is a highly conservative amino acid substitution and conformational changes caused by changing the connection between residues.(2)Cell function mechanism studies have confirmed that PRSS56 is a secreted protein and is located in Endoplasmic reticulum.In addition,the mutation does not change its subcellular localization.The concentration of serine protease secreted by the mutant transfected cells that cause myopia is higher than that of the high hyperopia group and the wild type,which is consistent with the loss of PRSS56 function leading to the reduction of the eyeball as well as the PRSS56 mutation associated with myopia.This preliminary prediction may work in the opposite way,consequently providing new insights for PRSS56 to regulate eye axis growth.(3)Transcriptome sequencing analysis results suggest that PRSS56 mutations cause high myopia regulatory pathways and microtubule cytoskeleton organization has a strong association.It may be related to the ANKHD1 gene,requiring to be verified in the next step.