The Location And Mutation Screen Of Pathogenic Gene Of Hereditary Aniridia,RNAi-Mediated PAX6Silencing And A Study In The Effect Of PAX6RNAi On B3Cells

Objective:We tried to identify the genetic defect causing hereditary aniridia in two families from Shandong Prov. and Henan Prov. of China; To construct vectors of RNA interference (RNAi) of the pathogenics gene PAX6; To study the effect of RNAi-mediated PAX6gene silencing on B3cell.Methods:1. Pathogenic gene screening:(1). To investigate a family affected by aniridia and cataract in Shandong and another family affected by aniridia in Henan, genomic DNA was extracted from peripheral blood leucocytes using standard protocols with informed consent. The affected status was determined before genetic analysis Determine the pattern of inheritance, exclude the evidence of chromosome defects in karyotype analysis.(2). To Shandong family, a genomewide scan consisting of382microsatellite markers spaced at-10cM intervals was performed using ABI PRISM Linkage Mapping sets. To Henan family, only several markers was selected in a candidate region based on the result of Shandong family, Genotyping and haplotype construction were performed;(3). Identify the potential mutation by sequencing the candidate gene.2. Construct PAX6RNAi vector:(1). Choose four PAX6candidate target sequences for RNAi, design corresponding shRNA sequences, construct and verification the recombinant plasmids. Construct293T cell model of exogenous expression of the PAX6fusion protein. Use shRNA plasmid vector for exogenous target sequence screening.(2). Construct shRNA lentiviral vector, and use it for endogenous sequence screening, and identify effective RNAi target sequence compared with the results of the exogenous screening.3. Infected B3cell with effective shRNA entiviral vector., observation the influence of the B3cell.Results:1.(1). Two families show an autosomal dominant pattern of inheritance by the analysis. There was no evidence of defects in karyotype analysis.(2). Haplotype analysis indicated that disease gene lay between D11S915and D11S4101in Shandong family, lay between D11S1755and D11S935in Henan family.(3) The candidate gene is PAX6. Sequencing of PAX6gene showed a heterozygous deletion A→/in intron2in the Shandong family, that may lead to a mistake when mRNA splicing, but we can’t find any abnormal change in the Henan family.2.(1). PAX6shRNA recombinant plasmid vector was successfully constructed. pDsRed2-Nl-PAX6fusion protein expression system was successfully successfully constructed. Exogenous expression model of the PAX6in293T cells was constructed. Exogenous screen target results show that the1st. the2nd and No.4target sequence is effective interference target point.(2). PAX6shRNA lentiviral vector was constructed successfully. Endogenous screen target results are consistent with exogenous screening results.3. PAX6silencing in B3cells lead that the number of apoptotic cells abnormal increase.Conclusions:1. A deletion mutation occurred (IVS2-2delA) in the PAX6second intron in Shandong family, resulting in the family appeared congenital aniridia and congenital cataract phenotype.2. In Henan aniridia family, the disease gene lay between D11S935and D11S1755, but has not found the specific gene.3. Find three effective RNAi target sequence.4. Construct PAX6RNAi plasmid vector and lentiviral vector.5. PAX6silence caused excessive apoptosis of B3cells.