Clinical Analyzing And Mutation Screening Of PAX6Gene In A Chinese Family With Congenital Aniridia Primary Culture, Subculture, Purification And Identification Of Human Dermal Fibroblasts

Objective Investigate the mutation of PAX6gene and its pathogenic implications in a Chinese family with congenital aniridia through clinical analyzing of the manifestations and mutation screening of the gene PAX6.Methods Clinical datas of five patients and two unaffected family members were collected and summarized in a pedigree to analyze the characteristics of the clinical manifestation. Genomic DNA of affected/unaffected family members mentioned before and one hundred unrelated normal controls was isolated from their peripheral blood, amplified in PCR with primers designed for fourteen exons of PAX6genes and sequenced with the method of Sanger. Localize the gene mutation by comparing the results of sequencing among patients, unaffected family members and unrelated normal controls, then investigate its pathogenic implications with bioinformatics.Results1.Five patients recruited had a common menifestation of iris defect while their ptosis and nystagmus are of various severities with corneal opacity and phacoscotasmus exist/absent, which diaplayed the phenotype diversity within family. The menifestations got worse with the age and was inhereted vertically and continously unrelated with the gender, revealing the autosomal dominant inheretance mode.2.Heterozygote mutation c.393-396delTAGC(p.Ser132LysfsX14) on exon seven was detected among five patients recruited but not the unaffected family members and unrelated normal controls. This mutation may result in the premature termination of open reading frame and the absence of Glu/Gly rich link domain, high conserved homeobox-DNA-binding domain and Pro/Ser/Thr-rich domain in the protein coded, thus leading to eye malformation due to PAX6haploinsufficiency.Conclusion The clinical menifestations of the congenital aniridia family were analyzed, during which we discovered that the severity of some menifestations can be inhereted and discussed the possible reasons. Heterozygote mutation c.393-396delTAGC (p.Ser132LysfsX14) on exon seven has been detected which was the basis of genetic counseling and prenatal diagnosis. This mutation has not been reported at home and aboard so the de novo mutation broadened the spectrum of PAX6gene mutation in Chinese. Objective Establish the methods of primary culture, subculture, purification and identification of human dermal fibroblasts in order to offer cells of sufficient purity and quantity for the following experiments.Methods Human dermal fibroblasts were primary cultured by outgrowing method from explanted tissue pieces, subcultured by trypsin digestion, and purified with differential digestion and adherence method. Observe the morphology and growth status of fibroblasts under inverted microscope. Stained the vimentin in purified fibroblasts with indirect immunofluorescence technique, identified and counted positive cells by fluorescence microscope.Results1.Fibroblasts outgrew from the edge of tissue pieces after one week or so since planted. They were adherent to the bottom of flask and of spindle or flat star shape at first while turned to swirl or palisade as time went by, and finally covered the entire bottom of25cm2culture flask at the end of4th week, roughly.2. Spindle fibroblasts coexisted with polygonal keratinocytes during primary culture and were purified effectively with differential digestion and adherence method after subculturing.3. Immunostained by rabbit vimentin primary antibody to human and FITC conjugated donkey anti-rabbit secondary antibody as well as DAPI, nuclei were stained blue and cytoskeleton green. Percentage of positive cells exceeded98%.Conclusion Human dermal fibroblasts were primary cultured successfully by outgrowing method from explanted tissue pieces and purified with differential digestion and adherence method, after which fibroblasts were qualified for the following experiments in purity and quantity.