The Role Of P-A Gene In Primary Congenital Glaucoma

Primary congenital glaucoma(PCG)is one of the hereditary ophthalmopathy which has been characterized as congenital trabecular meshwork and anterior chamber angle dysplasia.Symptoms associated with PCG include epiphora,photophobia,blepharospasm and buphthalmos.Patients with PCG exhibit corneal edema,increased corneal diameter,damaged Haab's striae or an enlarged axial length.Many of the PCG patients have abnormal eyes at birth.Onset of PCG is often during the first 3 years of life,while there are 80% patients suffered from PCG within their first year.Despite its rarity,PCG has a serious adverse effect on a patient's quality of life.Due to the limited understanding of the complex pathogenesis of PCG and the lack of effective interventions to prevent and treatment of PCG,it is particularly important to find new candidate genes associated with PCG.In the previous study,a new candidate gene P-A and its two mutations(c.580G>A,p.E194K;c.124-125>inscagcggggg,p.E42delinsAAAE)were identified in two PCG families by whole exome sequencing.This was also verified by Sanger sequencing.Based on the results of previous studies,this study would further explore the function of P-A gene,and clarifiy the role of P-A gene mutations in trabecular meshwork cells and the possible mechanism associated with primary congenital glaucoma.Methods:(1)Human trabecular meshwork cells(TM cell)were cultured.The TM cells were transfected with pCMV6-mutant P-A gene plasmid vectors(c.580G>A and c.124-125>inscagcggggg)respectively.The growth of TM cells was observed by optical microscope.(2)The gene expression was detected on mRNA level by RT-PCR and on protein level by Western Blot.(3)Immunocytochemistry was used to identify the co-localization of P-A gene in TM cells.(4)The expressions of GRP78,PDI and MMP-2 on mRNA level were conducted by RT-PCR.(5)The apoptosis of TM cells was observed by Hoechst33342 fluorescence staining by fluorescence microscope and AnnexinV-APC Flow Cytometry.(6)The adherence ability of TM cells was determined by CCK8.(7)The migration ability of TM cells was performed by wound healing experiment.Results:(1)P-A gene mutation influnced the normal shaps of TM cells.After mutation,the trabecular meshwork cells bacame narrow rhombus and the nucleus was larger than before.The cells were separated from each others,and the cell density was reduced significantly.(2)After transfection,the expression of P-A gene on mRNA and protein levels were decreased,while the gene expression in p.E194 K subgroup was decreased about 30% that of 15% in p.E42 delinsAAAE.(3)The abnormal accumulation of Ac45 protein on endoplasmic reticulum was observed by Immunocytochemistry.(4)In addition,the endoplasmic reticulum molecular chaperones GRP78 and PDI were increased both in two mutation groups.In the p.E194 K group,the expression of GRP78 and PDI was increased more than in p.E42 delinsAAAE group.The expression of MMP-2 on mRNA level was decreased both in p.E194 K and p.E42 delinsAAAE group.(5)p.E194 K mutation was lead to late phase apoptosis in TM cells and early apoptosis caused by p.E42 delinsAAAE mutation.(6)p.E194 K mutation inhibited the adherence ability of TM cells about 40.72% and 46.20% by p.E42 delinsAAAE.(7)p.E194 K mutation reduced the migration rate of trabecular meshwork cells by about 50%,while the migrate ability was lost by p.E42 delinsAAAE mutation.Conclusion: P-A gene plays an important role in the development and the normal functions of TM cells.Therefore,the study on P-A gene mutation in TM cells is helpful to illustrate the possible pathogenetics in PCG.