Identification And Functional Study Of Candidate Pathogenic P-X Gene For Primary Congenital Glaucoma

Primary congenital glaucoma is a kind of congenital atrial and angular dysplasia.Although surgery can be performed in patients during childhood,when their eyes are not fully developed,surgical scar is a big hidden danger in their growth process.Long-term drug treatment causes toxic side effects and has limited therapeutic value.At present,the most serious clinical manifestation of primary congenital glaucoma is the destruction of the optic nerve,which eventually leads to blindness.The damage of the optic nerve is irreversible,so it is particularly important to investigate the candidate pathogenic gene and its function of primary congenital glaucoma.Objective: To study the gene mutation of candidate pathogenic gene P-X for primary congenital glaucoma and the function of candidate pathogenic gene,and to explore the effect of P-X gene mutation on the pathogenesis of primary congenital glaucoma.Subjects: Three families of primary congenital glaucoma were studied.Methods: A total of 3 patients and their family members and 400 healthy controls who met the primary congenital glaucoma diagnostic criteria were collected.Peripheral blood genomic DNA was extracted for full exon sequencing and sanger sequencing verification,and bioinformatics analysis was performed.Mutation expression vector of P-X gene was constructed,and the function of candidate pathogenic genes including western blotting and cellular immunochemistry was conducted.Results: A candidate pathogenic mutation site c.580G>A(p.E194K)was found in both PCG-348 and PCG-35 families.This new mutation was missense in two patients with primary congenital glaucoma and their mother.Another candidate pathogenic mutation site c.124-125 > ins CAGCGGCGG(p.E42delinsAAAE),which was an insertion mutation in a patient with primary congenital glaucoma and his mother,was detected in PCG-424 families.The above two mutation points were not seen in 400 normal controls.By using SIFT online tool,it was found that the two new mutation points were pathogenic.Western blotting analysis showed that protein expression decreased about 50 percent with mutation of c.580G>A(p.E194K),and slightly decreased about 10 percent with mutation of c.124-125 > ins CAGCGGCGG(p.E42delinsAAAE).Cellular immunohistochemistry(ICC)showed thatthe protein encoded by P-X gene was mainly expressed on the cell membrane.The protein expression of P-X gene was decreased at the mutation point of c.580G>A(p.E194K),and the protein expression was down-regulated by the mutation of c.124-125 >ins CAGCGGCGG(p.E42delinsAAAE).The study in this paper showed that the mutation site of c.580G>A(p.E194K)may lead to the decrease of P-X protein synthesis,and the mutation of c.124-125 > ins CAGCGGCGG(p.E42delinsAAAE)probably resulted in the slight decrease of P-X protein synthesis.From the level of molecular biology,it is indicated that these two mutation sites of P-X are likely to be associated with PCG,and the new mutant points of candidate pathogenic gene expand the gene map information of P-X mutation with primary congenital glaucoma and provide certain theoretical reference basis for molecular diagnosis.