Screening And Functional Identification Of PRPF31 Gene Mutation In Non-syndromic Retinitis Pigmentosa Families

Background and AimsRetinal pigmentosa（RPI,OMIM: 268000）is a hereditary retinal disease caused by abnormalities of photoreceptor cells（including rod cells and cones）.The incidence rate of RP in the world is about 1 / 5000-1 / 3500,and the incidence in China is about l / l000 which is increasing year by year.It is one of the main reasons for blindness.RP is classified as syndromic RP and non-syndromic RP according to whether there are other symptoms.Non-syndromes RP（nonsyndromic retinitis pigmentosa,NSRP）accounts for about 65% of RP.The pathogenesis of RP is very complex,involving many different metabolic pathways.The proteins encoded by the pathogenic genes are mainly involved in the process of light conduction,the maintenance of the structure of photoreceptor cells and mRNA splicing.Mutations in the genes of various proteins in different biological pathways may lead to the degradation of the photoreceptor cells,which may lead to the occurrence of RP.In 2012,Xu Fei et al.found a c.544₆18del75bp new mutation on the 7th exon of the PRPF31 gene in an autosomal dominant RP family.However,the selected family in the study of Xu Fei et al.is not complete which is a small part of the wholefamily.RP has a strong clinical and genetic heterogeneity and there may be new pathogenic mutations which have not been found in the whole family.Inadequate exploration of the specific mechanism and function of the deletion mutation c.544₆18del75bp.So,we complement the family,and verify the results and search for new pathogenic mutations in the whole family.We try to further clarify the relationship between genotype and phenotype and study the function of the gene mutation to provide a theoretical basis for the study of RP pathogenesis and the future clinical diagnosis and treatment.SubjectsWe complemented the RP family of Xu Fei studies and collected the information of the disease history and ophthalmic examination of the family members and diagnosed the RP patients.Then we mapped the family pedigree after understanding the information of the family members.Extracted 5 ml of peripheral blood of a family member.Randomly selected 100 healthy individuals as the controls and extracted of peripheral blood 5ml.This study was approved by the Zhengzhou University Ethics Committee,and all participating individuals signed informed consent.Research methods1.The genomic DNA were extracted from the peripheral blood by extraction kit RNA was extracted from the RP family members and was reverse transcribed.2.The c.544₆18del75bp mutation of PRPF31 gene was confirmed by Sanger sequencing in all the families and 100 healthy controls.And identified the co-segregation of the genotype and phenotype in the family.Then tested the genomic cDNA of c.544₆18del75bp mutation in the RP patients by Sanger sequencing.3.The bioinformatics function was analyzed and the three-dimensional structure of protein was predicted by SWISS MODEL software.4.The real-time quantitative RT-PCR technique was used to detect the expression level of PRPF31 of the patients and normal subjects in this family.The wild-type and mutant PRPF31 gene overexpression vector was constructed andextracted by plasmid extraction kit.After transfection of 293 T cells,the expression of wild-type and mutant PRPF31 gene vector in 293 T cells was detected by Western blot.The mRNA expression of PRPF31 in 293 T cells transfected with wild-type and mutant overexpressing vectors was detected by RT-PCR.5.The genes such as RP9,ROM1,SNRNP200 and TOPORS,which were related to mRNA cleavage and expressed in human peripheral blood,were selected.The expression levels of these genes were detected by real-time quantitative RT-PCR in peripheral blood of the pediatric patients and normal subjects.The expression of these genes was verified in 293 T cells transfected with wild-type and mutant overexpressing vectors.6.Used SPSS19.0 software for data analysis.The level of mRNA expression between patients and healthy controls was statistically analyzed by Student’s t test.All quantitative data were expressed as mean ± SD.The Bivariate correlation analysis was used to analysis the correlation of the mRNA expression between the PRPF31 gene and its related genes of all members of the family.P<0.05 was statistically significant.Result:1.Most of the patients had the first symptom of the night blindness before 10 years old.The patients accompanied by decreased visual acuity and visual field defects.Fundus examination showed that the disc color is relatively normal.There are different degrees of retinal pigment cell atrophy.2.After sanger sequencing,both the DNA and cDNA of the patients were found to carry the c.544₆18del75bp mutation.In addition to a case of incomplete members,the family of normal and 100 cases of healthy control did not have the mutation.The results were consistent with the results of Xu Fei et al.In addition,we found a deletion mutation of IVS6-78_IVS6-75del4 CACA on the PRPF31 gene,but the mutation was not found in all patients.These two mutations were presented in one of the exogenous members of the family and were located on different chromosomes.The incidence of c.528-78₅28-5drCACA deletion mutation in normal family was31.4% and all of them were heterozygous mutations.38% of 100 healthy controls hadIVS6-78_IVS6-75del4 CACA mutations and were all heterozygous.The frequency of its allele was 21.5%.3.The three-dimensional structure of the protein encoded by the C.544₆18del75bp mutant PRPF31 gene predicted by SWISS MODEL.The results showed that the structure of the protein has changed obviously.Mutation Taster online software was used to predict the c.544₆18del75bp mutation site and IVS6-78_IVS6-75del4 CACA mutation site on PRPF31 gene.The results showed that c.544₆18del75bp deletion was a pathogenic mutation that could cause amino acid changes in the sequence changes in the cleavage site maight result in changes in the protein structure.While the IVS6-78_IVS6-75del4 CACA site maight alter the cleavage site and the structure of the protein.4.The mRNA expression of PRPF31 was detected by real-time quantitative RT-PCR in 16 patients and 26 normal controls.It was found that the mRNA expression level of PRPF31 gene in patients（0.65 ± 0.40）was significantly lower than that in the normal controls（1.35 ± 1.15）（P <0.05）.The overexpression vector of wild type and mutant PRPF31 gene was transfected into 293 T cells.The expression of PRPF31 overexpression vector in 293 T cells was detected by Western blot.The mRNA expression level of PRPF31 gene in wild-type and mutant PRPF31 gene transfection group was significantly higher than that in negative control group（p<0.001）,and the mRNA expression level of PRPF31 gene in wild type and mutant PRPF31 gene was significantly higher than that in negative control group（P<0.001）.5.The mRNA expression levels of RP9,ROM1,SNRNP200 and TOPORS were detected by real-time quantitative RT-PCR in 16 patients and 26 normal controls.The mRNA expression levels of RP9 and ROM1（0.52 ± 0.34 and 0.79 ± 0.67,respectively）were significantly lower than those of normal controls（1.50 ± 1.13 and1.74 ± 1.72,respectively）（P <0.05）.Association study of the mRNA expression level of PRPF31 gene in 39 patients（16 patients and 26 normal controls）and the expression level of RP9 or ROM1 gene showed that PRPF31 gene was a significant positive correlation with mRNA expression of RP9 gene（r = 0.71,P = 0.000）.The expression levels of RP9,ROM1,SNRNP200 and TOPORS genes in transfected 293 T cells were verified and found that the expression level of RP9 in mutant PRPF31 transfection group was lower than that in wild type transfection group,but the difference was not statistically significant.Conclusion1.The heterozygous mutation c.544₆18del75bp on the PRPF31 gene may be the pathogenic mutation in this retinitis pigmentosa family and the IVS6-78_IVS6-75del4 CACA deletion mutation may be a polymorphic loci.2.c.544₆18del75bp mutation of PRPF31 gen can reduce the normal expression of the gene in the patient,which the important mechanism of this retinitis pigmentosa family.3.The c.544₆18del75bp mutation on the PRPF31 gene could significantly decrease the expression level of ADRP-related gene RP9,which indicated that the c.544₆18del75bp mutation of PRPF31 gene could lead to the occurrence of RP by a variety of channels.