摘要目的 探讨1个Waardenburg综合征(Waardenburg syndrome,WS)2型家系的分子致病机制.方法 对WS2型家系成员进行MITF、SOX10和SNAI2基因的突变检测,对发现的新突变进一步验证,并构建MITF基因及其突变体表达质粒,分别瞬时转染黑色素瘤UACC903细胞,应用Western印迹和细胞免疫荧光法检测MITF野生/突变蛋白在UACC903黑色素瘤细胞中的表达和亚细胞分布.结果 测序结果显示患者MITF基因第8外显子存在c.763C＞T(p.R255X)杂合突变,在家系内与疾病共分离,为已报道致病性突变.未发现SOX10和SNAI2基因的致病性突变.MITFw11d及其突变体MITFR255x表达质粒经DNA测序鉴定序列正确,两者均在UACC903细胞中正确表达,MITFwi1d仅在细胞核中分布,MITFR255x在细胞质与细胞核中均有分布.结论 MITF基因c.763C＞T(p.R255X)突变是导致该WS2型大家系发病的分子病因,该突变对MITF蛋白的亚细胞定位产生影响,为在体外实验进一步研究MITF基因突变致WS发病的分子机制奠定了实验基础.

abstractsObjective To explore the pathogenetic mechanism of a family affected with Waardenburg syndrome.Methods Clinical data of the family was collected.Potential mutation of the MITF,SOX10 and SNAI2 genes were screened.Plasmids for wild type (WT) and mutant MITF proteins were constructed to determine their exogenous expression and subcellular distribution by Western blotting and immunofluorescence assay,respectively.Results A heterozygous c.763C＞T (p.R255X) mutation was detected in exon 8 of the MITF gene in the proband and all other patients from the family.No pathological mutation of the SOX10 and SNAI2 genes was detected.The DNA sequences of plasmids of MITFwild and mutant MITFR255x were confirmed.Both proteins were detected with the expected size.WT MITF protein only localized in the nucleus,whereas R255X protein showed aberrant localization in the nucleus as well as the cytoplasm.Conclusion The c.763C＞T mutation of the MITF gene probably underlies the disease in this family.The mutation can affect the subcellular distribution of MITF proteins in vitro,which may shed light on the molecular mechanism of Waardenburg syndrome caused by mutations of the MITF gene.