Molecular Genetics Study On The Pathogenic Mechanism Of Congenital Cataract

Objective: To study the pathogenesis of congenital cataract from molecular biology and molecular genetics,and to explore the intervention of cataract formation mechanism.Methods: According to different phenotypes of patients,different screening and research methods were selected.For families with hereditary cataracts and families with sporadic cataracts,the panel covering all exon regions of 4813 clinically relevant genes or whole exon sequencing(WES)was selected to screen for suspected nucleotide variations.In 10 trio families with congenital cataract and congenital heart disease,we performed whole exon sequencing(WES)and comparative genomic hybridization(a CGH)to screen for suspected nucleotide variations and large fragment copy number variations.The new mutations of GJA8,CRYBA1,and GALK genes were found in hereditary cataract families,and two of them,GJA8 and CRYBA1,were evaluated for functional verification and mechanism study.The pEGFP-N1 overexpressed plasmid of wild-type and mutant-type GJA8 and p3×Flag-CMV-14 overexpressed plasmid of wild-type and mutant-type CRYBA1 were constructed to study the subcellular localization,aggregation formation,solubility change,cell proliferation and apoptosis of wild-type and mutant-type protein.In this study,it was found that the pathogenic mechanism of CRYBA1 gene mutation was the formation of lens protein aggregates.According to the reported studies,lanosterol can reverse lens protein aggregates in cataracts.Therefore,the lanosterol dissolution experiment was conducted on lens protein aggregates formed by CRYBA1 gene found in this study,and the dissolution effect of lanosterol on different types of lens protein aggregates was discussed.Results: In 6 families with hereditary cataracts and 2 trio families with simple sporadic cataracts,we found 8 mutation sites of congenital cataracts.2 pathogenicity chromosomal variations and 1 gene mutation site were identified in the trio family genetic analysis of 10 cases of congenital cataract with congenital heart disease.Four new mutations were identified,including GJA8(c.669G>C,p.E223D)in Family1,CRYBA1(c.591 dup G,p.E197fs)in Family 2,GALK(c.1159G>A,p.A387T)in Family 5 and GJA8(c.590C>T,p.S197F)in Family 11.These genes and chromosomal variations were analyzed and discussed.We validated the function of the new mutations in Family 1 and Family 2 whose patients had a simple cataract phenotype and no other systemic diseases.E223D-CX50 was found to be incorrectly localized to the ER and induced apoptosis.The E197fs-CRYBA3 protein forms aggregates in the nucleus and its solubility is reduced.Then we used lanosterol to dissolve E197fs-CRYBA3 protein and found that it had a certain solubility to the protein.Conclusion: This study once again proves that congenital cataract has genetic heterogeneity.The pathogenesis of congenital cataract with congenital heart disease was analyzed.The pathogenesis of hereditary cataract induced by GJA8(c.669G>C,p.E223D)and CRYBA1(c.591 dup G,p.E197fs)was preliminarily verified.Lanosterol has been shown to partially dissolve the accumulated lens proteins in lens cells in vitro,which provides an idea for the treatment of cataract with drugs.