Genetic Screening Of An RP Family And The Study Of IPS-RPE Cells Of Patients

Part Ⅰ : Clinical and genetic analysis of a family with autosomal dominant retinitis pigmentosaObjective:To investigate the clinical manifestations and genetic condition of a family with autosome dominant retinitis pigmentosa(ADRP),Methods:Detailed clinical investigations,the family tree was pictured.Genomic DNA was extracted from peripheral blood of 12 participants by standard phenol extraction protocols.All participants underwent ophthalmic examinations.Targeted sequence capture array technique with next-generation of high throughput sequencing(NGS)was performed to detect variants in 179 identified hereditary retinal disease(HRD)causing genes and 10 potential causative genes.Variants detected by targeted sequencing were filtered by bioinformatic analyses.Results:There are 12 members in the four generations family,Peripheral blood samples from 12 individuals were collected.5 patients were distributed in three successive generations,and the gender difference was no significant,from which a autosomal dominant inheritance characteristic was demonstrated.we identified a mutation gene(SNRNP200p.S1087L)in this family.Clinical features contained early onset age,rapid disease progression.This family demonstrated tpically night blindness and bone spicule pigmentation.Conclusions:we identified SNRNP200p.S1087L as a hotspot mutation in the present family.The distinct phenotypes including early onset age,rapid disease progression and severely impaired visual function.Part Ⅱ: Research on induction of retinitis pigmentosa patient iPS cells from human dermal fibroblasts and induction of iPS cells to retinal pigment epithelium cells.Objective:To induce a cell line of point mutation SNRNP200p.S1087L of retinitis pigmentosa patient-specific iPS cells by retroviral methods and verify their treatment potential.iPS cells were differentiated into retinal pigment epithelial cells.Methods:1 patients’ skin fibroblasts were cultured by trypsin digestion,The fibroblasts were transfected by a series of retrovirus to generate iPS cells.iPS cells and iPS RPE cells obtained were identified by cell morphology,alkaline phosphatase staining,the pluripotent stem cell surface marker stainin.Results:Successfully isolated fibroblasts cells from the skin of patients,and the fibroblasts cells can be subcultured.Successfully induced and identified RP patient-specific iPS cells,the iPS cells express induced plufipotent stem cell surface markers,successfully differentiated iPS cells into retinal pigment epithelial cells,providing a theoretical basis for future cellar and gene therapy for RP.Conclusions:Point mutation SNRNP200p.S 1087L of retinitis pigmentosa patient-specific iPS cells can be obtained from the human dermal fibroblast,and can be differentiated into retinal pigment epithelial cells.