CAJ | 학술논문

目的：探讨配对盒基因（pair box 3，PAX3）突变对小眼球畸形相关转录因子（microphthalmia－asso‐ciated transcription factor ，MITF）基因转录活性的影响及其在I型Waardenburg综合征（Waardenburg syndrome ， WS）发病中的作用。方法野生型PAX3及其致病突变H80D和H186fsX5表达质粒瞬时转染293T细胞，应用荧光素酶活性检测系统对M IT F报告基因活性检测，观察野生／突变 PAX3蛋白对其靶基因M IT F转录活性的调控作用及二个突变蛋白对野生PAX3蛋白功能的影响；应用生物素标记的含序列attaat的DNA寡核苷酸链探针分别沉淀PAX3、H80D和H186fsX5蛋白，检测野生／突变PAX3蛋白与靶基因MITF启动子的结合力。结果尽管H80D蛋白仍残余部分功能可增加M IT F启动子转录活性，但与野生PAX3蛋白相比，二者差异有显著统计学意义（P＜0．01），而 H186fsX5蛋白则完全失去调控MITF启动子转录活性作用（P＜0．01）；二者均未对野生PAX3蛋白功能产生显性负效应作用（P＞0．05）。突变 H80D蛋白与野生 PAX3蛋白均可与MITF启动子特异DNA序列attaat结合，而突变H186fsX5蛋白则不能与之结合。结论 H80D和H186fsX5影响靶基因M IT F转录活性，使其表达下调、黑色素合成减少，以单倍体剂量不足效应致I型WS。
목적：탐토배대합기인（pair box 3，PAX3）돌변대소안구기형상관전록인자（microphthalmia－asso‐ciated transcription factor ，MITF）기인전록활성적영향급기재I형Waardenburg종합정（Waardenburg syndrome ， WS）발병중적작용。방법야생형PAX3급기치병돌변H80D화H186fsX5표체질립순시전염293T세포，응용형광소매활성검측계통대M IT F보고기인활성검측，관찰야생／돌변 PAX3단백대기파기인M IT F전록활성적조공작용급이개돌변단백대야생PAX3단백공능적영향；응용생물소표기적함서렬attaat적DNA과핵감산련탐침분별침정PAX3、H80D화H186fsX5단백，검측야생／돌변PAX3단백여파기인MITF계동자적결합력。결과진관H80D단백잉잔여부분공능가증가M IT F계동자전록활성，단여야생PAX3단백상비，이자차이유현저통계학의의（P＜0．01），이 H186fsX5단백칙완전실거조공MITF계동자전록활성작용（P＜0．01）；이자균미대야생PAX3단백공능산생현성부효응작용（P＞0．05）。돌변 H80D단백여야생 PAX3단백균가여MITF계동자특이DNA서렬attaat결합，이돌변H186fsX5단백칙불능여지결합。결론 H80D화H186fsX5영향파기인M IT F전록활성，사기표체하조、흑색소합성감소，이단배체제량불족효응치I형WS。
Objective To investigate the impact of pair box 3 (PAX3) gene mutations on transcriptional ac‐tivity of target gene microphthalmia -associated transcription factor (MITF) and the role it plays in the pathogene‐sis of Waardenburg syndrome type I .Methods The 293T cells were transient transfected with wild type (WT ) PAX3 and mutant type (M T ) H80D ,H186fsX5 plasmids .We observed and analysed the regulation effects of WT/MT PAX3 on the transcriptional activities of MITF and the influence of the two mutants on WT PAX3 function u‐sing luciferase activity assays ,detect DNA binding capacity of WT/MT PAX3 to MITF gene promoter using a bioti‐nylated double - stranded oligonucleotide probe containing PAX3 binding motif ATTAAT to precipitate PAX3 , H80D and H186fsX5 respectively .Results H80D mutant was partially functional and was able to transactivate the MITF promoter in part ,but it was dramatically reduced as compared with WT PAX 3(P<0 .01) .H186fsX5 mu‐tant was loss -of -function and failed to transactivate the MITF promoter as compared with WT PAX 3 (P<0 .01) . None of them showed dominant -negative effect on WT PAX3(P>0 .05) .WT PAX3 and H80D mutant were able to bind specifically to the ATTAAT motif on the MITF promoter ,whereas H186fsX5 PAX3 lost the DNA -binding ability .Conclusion The mutations H80D and H186fsX5 made down-regulation of MITF transcription and decrease syn‐thesis of melanin ,which resulted in haploinsufficiency of PAX3 protein and caused mild phenotypes of WS1 .