Investigation Of ADAR1 Gene Function And Its Role In DSH By Using Zebrafish Model

Objective : In this study,we aim to investigate the influence of ADAR1 gene on DSH,whether there is cell apoptosis or c-kit gene expressive difference between hyperpigmented and hypo-pigmented areas,and know the pathogenesis of DSH.Method:?1?To perform mutation analysis of ADAR1 gene in an indexed DSH pedigree?2? To determin the function of adar1 in zebrafish development,we utilized the morpholino to knockdown the expression of adar1 in zebrafish embryos and observed the phenotypes of adar1 insufficiency in the 52 hpf and 76 hpf of embryo development.?3?Zebrafish were photographed at 48 and 72 hpf under a stereo microscope coupled with a charge-coupled device for obtaining quantitative information on melanin contents.?4?Fifty embryos per sample were collected at 8,16,28,32,36,52,64,and 76 hpf for RT-PCR assessment of the time course of adar1 expression.?5?We utilized quantitative RT-PCR to determine the expression levels of a adar1 transcripts in adult zebrafish tissues and also quantify the relative m RNA expressions of c-kit,mitf,wnt11,cdc42,rhoa in 48 and 72 hpf embryos.?6? The difference in the expression of C-kit between Dsh patients hyperpigmented and hypo-pigmented areas were dected by means of immunohistochemical methods.?7? The apoptosis condition between Dsh patients hyperpigmented and hypo-pigmented areas were analyzed by using tunel assay.Results:?1?A frameshift mutation c.1019₁020del AG?p.Gln340Argfs*5? in exon 2 of ADAR1 gene was indentified in the affected members of this DSH pedigree?2?Adar1 was successfully knocked down in adar1-mo group as compared with sc MO group and untreated group.?3? The melanin contents in the adar1-mo zebrafish tail at 48 hpf was the same to sc MO and untreated control.The melanin contents in the adar1-mo zebrafish tail at 72 hpf increased significantly compared with sc MO and untreated control groups.?4? Morpholino knockdown of adar1 in zebrafish produced a phenotype characteristic of melanocyte cell polarity change at 48 and 72 hpf.Morpholino knockdown of adar1 in zebrafish produced a phenotype characteristic of melanocyte abnormal migration and distribution at 72 hpf.?5? Knockdown of adar1 in zebafish reduces the expression of rhoa?cdc42 at 48 hpf. Knockdown of adar1 in zebafish reduces the expression of wnt11 at 72 hpf. Knockdown of adar1 in zebafish reduces the expression of c-kit? mitf at 48 and 72 hpf.?6?The expression of C-kit was higher in hyperpigmented area than hypopigmented one.?7? More apoptosis was noted in hypopigmented area than hyperpigmented one.Conclusion: A novel pathogenic mutation c.1019₁020del AG ?p.Gln340Argfs*5? was indentified via mutation analysis in this DSH pedigree.The study with adar1 knock-down zebrafish model sheds new sight on the effect to the melanocyte migration and cell polarity from gene adar1 mutation, which provides a novel rationale to know the pathogenesis of DSH.