The Investigation Of Molecular Mechanism Of Waardenburg Syndrome Type Two

Background:Waardenburg syndrome (WS) is the most common type of syndromic hereditry hearing loss, which is manifestated of pigmentation anomalies and hearing loss. It is always autosomal dominant, with a high degree of genetic heterogeneity. WS can be divided into four types:WS1, WS2, WS3and WS4. WS2is the most common one. Its pathogenic genes are MITF, SOX10and SNAI2. MITF accounts for about15%of WS2patients, as well as SOX10. However, the other70%of WS2patients are left unknown, and the molecular mechanism that how the genotype leads to the phenotype is unclear.Objective and Methods:In order to further investigate the molecular mechanism of WS2, the following plans were applied:1. The WS2families were continued to be collected, with a detailed analysis of phenotype and mutation screening of the MITF, SOX10and SNAI2.2. Western blot to study protein expression and immunofluorescence assay to investigate subcellular distribution, after constuction of eukaryotic expression plasmids and transient transfection into melanoma UACC903cells.3. To clarify the drosophila Mitf gene fuction, observe the phenotype of F1flies after hybrizating the GMR and DA GAL4lines with the UAS Mitf RNAi lines.Results:1. Eight WS2families were collected altogether. Three of them were pedigrees, the left were sporadic cases. Two heterozygous mutations, c.763C>T (R255X) and c.328C>T (R110X), were found in two pedigrees, which were proved to be cosegregation at last. The pathogenic factors of remaining six families were not found. R255X mutation has been reported in a four-generation Chinese WS family, while R110X mutation was found in two WS families.2. The pCMV-R255X-Flag mutant plasmid was successfully constructed and transfected into UACC903melanoma cells, and we found that the mutant protein was significantly reduced, which confirmed that the MITF R255X mutation encoded a truncated protein. Immunofluorescence assay indicated that the wild-type MITF protein is mainly distributed in the nucleus, while the R255X protein located in both the nucleus and the cytoplasm.3. Hybridizating GMR-GAL4and Da-GAL4lines with UAS-MITF RNAi lines, the F1generation’s phenotype were no significant difference with the control group.Conclusions:1. The MITF gene R255X and R110X mutation are hot spots of the Chinese population, while the left75%WS2families remain unknown. The result suggests that there are unknown WS genes to be discovered.2. R255X mutant protein due to loss of part HLH and all Zip area can not play the full function. Right subcellular location is also important for functioning.3. The drosophila Mitf gene expression is time and tissue specific in the development. UAS-Mitf RNAi flies need corresponding GAL4lines to drive the target gene expression.