A c.544_618del75bp mutation in the splicing factor gene PRPF31 is involved in non-syndromic retinitis pigmentosa by reducing the level of mRNA expression
Figure 1. Pedigrees of Chinese families with autosomal dominant retinitis pigmentosa. (A)Pedigree of previous studies by Fei Xu et al. Arrow indicate the proband. (B)Supplementary ADRP pedigree in this study. Figure 2. Clinical images an ERG examination data of the proband. (A, B) The proband's fundus images showed that the patient had pigmentation on the fundus and the blood vessels became thinner. (C)Proband ERG examination showed that a wave b wave disappeared. Figure 3. Identification of the PRPF31 gene in family. (A)Sanger sequencing results of c.544_618del75bp site in genomic DNA. (B)Sanger sequencing results of the IVS6-78_IVS6-75del4CACA deletion site in genomic DNA. (C)Sanger sequencing results of the c.544_618del75bp site in genomic cDNA. Figure 4. SWISS MODEL predicts the three-dimensional structure of the protein encoded by the mutated PRPF31 gene. (A)Wild-type PRPF31 gene-encoded protein. (B)Mutant PRPF31 gene-encoded protein. Figure 5. Effect of PRPF31 gene c.544_618del75bp deletion mutation and other ADRP pathogenic genes on mRNA expression. (A)Relative expression level of PRPF31 gene mRNA between patients in the family and normal controls, P*<0.05. (B)Relative expression levels of RP9, ROM1, SNRNP200, and TOPORS gene mRNAs between patients in the family and normal controls. P*＜0.05,P**＜0.01. (C)Correlation analysis between PRPF31 gene and RP9 gene mRNA expression level, r=0.7315，P<0.001. Figure 6. In vitro functional verification of c.544_618del75 bp mutation of PRPF31 gene and other ADRP pathogenic genes. (A)Relative expression levels of PRPF31 gene mRNA in 293T cells of four experimental groups, P＜0.001. (B)Expression of 293T cells transfected with overexpression vector carrying wild-type and mutant PRPF31 genes for 24 h. VEC served the empty expression vector as a negative control. (C)Protein expression analysis of WT and mutant PRPF31 revealed by western blotting. P*＜0.05,P**＜0.01. (D)Relative expression levels of mRNA of RP9, ROM1, SNRNP200 and TOPORS genes in 293T cells of four experimental groups. Table 1. Members of the family PRPF31 gene mutation site Sanger sequencing results. Table 2. 100 healthy controls PRPF31 gene mutation site Sanger sequencing results.
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1.Subjects recruitment and Clinical assessment. This study was approved by the Zhengzhou University Ethics Committee, and all participating individuals signed informed consents. 2.Sanger Sequencing to DNA and cDNA 3.Function Prediction NCBI database (http:/7www.ncbi.nlm.nih.gov/pubmed/);The Human Protein Atlas database (http://www.proteinatlas.org/); SWISS MODEL(https://www.swissmodel.org). 4.Real-time PCR analysis 5.Cell culture and Plasmids 6.Western blot Analysis