| Deafness is one of the most common clinical hereditary diseases.The common pathogenic etiologies of hearing loss including genetic causes and environmental factors,more than 60-80%of individuals can be attributed to genetic causes and about 20-40%of cases are caused by environmental factors.Most of Hereditary hearing loss,caused by mutations in a single gene,follows the law of Mendelian inheritance.In our previous study,using exome sequencing approach,we identified one novel deafness gene WYF1,a novel causative nonsense mutation p.E375X in DFNA5,and three novel pathogenic mutations p.L517P,p.I1967del,and p.LTV1557-9del in OTOF.Currently,with the CRISPR/Cas9 genome editing system,we created the mutated zebrafish model of wyflb and dfna5b,and some functional studies have been performed to explore the pathogenic mechanism of the deafness genes.Part I Creation of the Zebrafish Model of wyfb and its Functional StudiesThe heterozygous framshift mutation p.D326AfsX42 in WYF1 segeregated with the deafness phenotype in affected subjects.WYF1 is a transcriptional factor,expressed in the otic vesicle and optic vesicle of mouse,chick and zebrafish.With the CRISPR/Cas9 genome editing system,we created the mutated zebrafish model of wyflb,and the mutated locations are similar with the mutation site identified in humans,both locating at Exon 5.The survival rate and reproduction ability of wyflb mutated zebrafish are significantly lower than the wild type,and some individuals presented with abnormal development of heart.Fused otoliths were observed in some mutated zebrafish and morphants.The results of righting reflex and vestibular-ocular reflex tests show that the vestibular function of the wyflb homozygotes and heterozygotes are abnormal.Besides,the preliminary C-startle response results suggested that the mutated zebrafish larve might have heaing disability.There are no significant differences of the number and morphology of hair cells and the kinocilium and cilium between the wild and mutated zebrafish,implicating that the mutated Wyflb might not influence the development of hair cells.Part II Creation of the Zebrafish Model of dfna5b and its Functional ExplorationThe p.E375X mutation we found is the first pathogenic mutation in exon 8.This finding will play an important role in the exploration of the pathogenic mechanism of DFNA5.With the CRISPR/Cas9 genome editing system,we created the mutated zebrafish model of dfna5b(p.T328X and p.T328NfsX7),and the p.T328X mutation is more similar with the mutation p.E375X identified in humans.The development of dfna5b mutated zebrafish in every phase was quite normal,and no significant abnormalities were observed in the inner ear.Functional studies performed on of the mutated and wild type zebrafish are quite normal.There are no significant differences of the number and structure of hair cells between the wild and mutated zebrafish. |
