SOX10基因突变致Ⅱ型Waardenburg综合征发病的实验研究  

Molecular pathogenesis of Waardenburg syndrome type H resulting from SOXIO gene mutation

作　　者：张华[1,2] 陈红胜[3] 冯永[3] 钱敏飞[2] 李吉平[2] 刘君[2] 张淳[2] 

机构地区：[1]新疆医科大学第一附属医院耳鼻咽喉科,乌鲁木齐830011 [2]上海交通大学医学院附属仁济医院耳鼻咽喉科,200127 [3]中南大学湘雅医院耳鼻咽喉科,长沙410008

出　　处：《中华医学遗传学杂志》2016年第4期466-470,共5页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金（81260160,81500803）

摘　　要：目的通过体外实验探讨SOXl0基因突变E248fs致Ⅱ型Waardenburg综合征（Waardenburgsyndrome，WS）发病的分子机制。方法野生SOX10及其致病突变E248fs表达质粒瞬时转染293T细胞，用荧光素酶活性检测系统观察野生／突变SOXIO蛋白对其靶基因MITF转录活性的调控作用以及突变蛋白对野生SOXIO蛋白功能的影响；用生物素标记的含序列eattgtc的DNA寡核苷酸链探针分别沉淀SOX10和E248fs蛋白，检测野生／突变SOXIO蛋白与靶基因MjTF启动子的结合力；免疫共沉淀检测观察并分析SOXIO和E248fs蛋白稳定性变化。结果E248fs突变蛋白完全失去调控MjTF启动子转录活性作用（P〈0．01），并对野生SOXIO蛋白功能产生显性负效应作用（P〈0．05），其与野生SOXIO蛋白均可与MJTF启动子特异DNA序列cattgtc结合，但较野生SOXIO蛋白衰减加快。结论尽管E248fs具有显性负效应，但其稳定性降低，使得其影响靶基因MITF转录活性，导致黑色素合成减少，最终以单倍体剂量不足效应致Ⅱ型WS。Objective To explore the molecular mechanism of Waardenburg syndrome type Ⅱ （WS2） resulting from SOXIO gene mutation E248fs through in vitro experiment. Methods 293T cells were transiently transfected with wild type （WT） SOX10 and mutant type （MT） E248fs plasmids. The regulatory effect of WT/MT SOX10 on the transcriptional activity of MITF gene and influence of E248fs on WT SOX10 function were determined with a luciferase activity assay. The DNA binding capacity of the WT/ MT SOX10 with the promoter of the MITF gene was determined with a biotinylated double-stranded oligonucleotide probe containing the SOX10 binding sequence cattgtc to precipitate MITF and E248fs, respectively. The stability of SOX10 and E248fs were also analyzed. Results As a loss-oh{unction mutation, the E248fs mutant failed to transactivate the MITF promoter as compared with the WT SOX10 （P〈0. 01）, which also showed a dominant-negative effect on WT SOXl0. The WT SOXl0 and E248fs mutant were also able to hind specifically to the cattgtc motif in the MITF promoter, whereas E248fs had degraded faster than WT SOXl0. Conclusion Despite the fact that the E248fs has a dominant-negative effect on SOX10, its reduced stability may down-regulate the transcription of MITF and decrease the synthesis of melanin, which may result in haploinsufficiency of SOX10 protein and cause the milder WS2 phenotype.

关 键 词：WAARDENBURG综合征 SOX10基因 MITF基因 基因突变 体外实验 

分 类 号：R596.1[医药卫生—内科学]