Studies Of Molecular Mechanism Of Waardenburg Syndrome Caused By Mutations Of MITF, PAX3, And SOX10Genes

Waardenburg syndrome (WS) is the most common autosomal dominant inherited disorder in syndromic hearing loss and is classified into four types (WS1-4) depending on clinical features. There is no intrinsic causal fixed relationship between genotype and phenotype due to incomplete penetrance. The study of WS in the Chinese population is at the genomic level study including the family and sporadic survey of a small sample and mutations detection of related genes and MITF, PAX3, and SOX10are mainly causative genes. In the previous studies, we analyzed the clinical features of22WS patients, including9cases in4pedigrees and13sporadic cases, came from the central south in China, in which5were diagnosed as WS1,15as WS2,and2as WS4. At the molecular level,totally9novel heterozygous mutations (R217I and T192fs in MITF resulting in WS2, H80D and H186fs in PAX3resulting in WS1,G37fs, G38fs, R43X, and E248fs in SOX10resulting in WS2, W85X in SOX10resulting in WS4) were identified. How these mutations cause WS has been remained unclear so far.For further study the molecular mechanism of WS, we performed a series of in vitro studies on the nine mutations of the MITF, PAX3, and SOX10genes, including generation of eukaryotic expression plasmids of mutation genes, Western blot assays to detect mutant proteins expression, luciferase activity assays to detect mutant proteins activities, immunofluorescence assays to observe mutant proteins subcellular localization, co-immunprecipitation assays to detect interaction between mutant proteins and wild type (WT) proteins as well as mutant protein binding to DNA, in order to understand functional consequences of these mutations and its impact on WT proteins at the level of transcription and post-transcription. The results showed that the function of mutant proteins resulting from nine heterozygous mutations was altered. All WT and mutant proteins produces by eukaryotic expression plasmids were detected at the expected size in293FT cells. All mutants were loss of function and were not able to transactivate the transcriptional activities of target genes in Luciferase activity assays except R217I MITF and H80D PAX3, which were partially functional. All mutants failed to affect the activities of WT proteins in a dose-depandent manner except E248fs SOX10showing dominant negative effect. In immunofluorescence assays, R217I MITF, H80D PAX3, and E248fs SOX10were localized only in the nucleus in NIH3T3cells as the same as WT proteins did, whereas other mutants showed aberrant subcelluar localization both in the nucleus and cytoplasm, in which the distribution of H186fs PAX3and G38fs SOX10were mainly localized within the cytoplasm and the distribution of T192fs MITF and SOX10mutant G37fs, R43X, and W85X were mainly localized within the nucleus. Though both H80D PAX3and H186fs PAX3were able to interact with WT SOX10in co-immunoprecipation assays, they lost synergistic effect on WT SOX10to upregulate the expression of MITF in luciferase activity assays. All SOX10mutants lost synergistic effect on WT PAX3to unregulated the expression of MITF and all of them except E248fs were not able to interact with WT PAX3. E248fs SOX10can bind to DNA on the MITF promoter, which further demonstrate its dominant negative effect on WT SOX10, however, it degraded significantly faster than WT one. We draw conclusions from the results in these studies:(1) Though R217I MITF, being partially functional, showed the same subcelluar localization with WT MITF protein, it does not reach the threshold level enough for the fully normal function of WT MITF protein. T192fs MITF is loss-of-function due to its lacking of functional domains and aberrant subcellular localization. Two MITF mutants affect the development of melanocytes through directly downregulating the expression of tyrosinase.(2) H80D PAX3is partially functional and shows the same subcelluar localization with WT PAX3protein, whereas H186fs PAX3is loss of function due to its lacking of part functional domains and aberrant subcelluar localization. The two PAX3mutants fail to be in synergy with WT SOX10and downregulate the expression of MITF.(3) SOX10truncated proteins resulting from G37fs, G38fs, R43X, and W85X, lacking all functional domains, are loss of function and failure to transactivate, alone or in synergy with WT PAX3, the transcriptional activity of MITF. Though E248fs results in a truncated protein which still retains the functional domain binding to DNA and shows a dominant negative effect to inhibit function of WT SOX10, it functions equivalently as a null mutation for its faster decay than WT SOX10. It is the first time to perform a preliminary study on the molecular mechanism of pathogenesis of WS2caused by mutations of SOX10.(4)The results of these studies show that halpoinsufficiency underlies the mechanism leading to WS caused by the nine heterozygous mutations, which result in mutant proteins functioning closely related to their structure of the functional domain. The dosage of mutant proteins does not reach the threshold level necessary for the protein to exert their normal function and make melanocytes achieve normal development. The mutations of MITF, directly downregulating the expression of tyrosinase, and the mutations of PAX3or SOX10, directly downregulating the expression of MITF, affect the development of melanocytes and further lead to abnormality of the biosynthesis, transportation, and distribution of melanin in melanocytes, which leads to WS due to lacking melanin in iris, skin, and inner ear.(5) We reveal and elaborate the pathogenesis of WS in Chinese population at molecular and celluar level through these in vitro studies, which established a more comprehensively experimental system for functional test of causative mutations resulting in WS and lay experiment theory and basis for further study of the pathogenesis of WS at animal level in vivo.