Ⅱ型Waardenburg综合征的分子机制研究

发布时间：2019-06-26 15:04

【摘要】：研究背景：Waardenburg综合征(Waardenburg Syndrome,WS)是以听力障碍和色素分布异常为主要表现的遗传综合征,以常染色体显性遗传为主,具有高度的遗传异质性。WS根据临床表型不同,可以分为四型：WS1、WS2、WS3、WS4。其中WS2是最常见的一种类型,目前发现致病基因有MITF、SOX10和SNAI2,MITF和SOX10约各占所有WS2病人致病原因的15%,仍有70%的WS2病人未找到致病因素,且基因突变如何导致表型发生的分子机制尚不清楚。目的和方法：为了进一步探讨WS2患者的基因型与表型的内在联系,本研究通过以下方案探讨WS的分子机制：1.收集WS2家系,采集每个患者的详细临床资料,并对相关致病基因MITF、SOX10、 SNAI2的外显子进行突变筛查。2.对发现的MITF基因R255X突变,构建真核表达质粒pCMV-R255X-Flag,瞬时转染黑色素瘤细胞,Western Blot验证突变蛋白表达量,免疫荧光检测突变蛋白的亚细胞分布情况。3.将Mitf RNAi品系果蝇与GAL4品系果蝇杂交,观察子一代果蝇的表型。结果：1.总共收集到3个WS2家系,5个散发病例。并在其中的两个大家系中分别发现MITF基因的c.763CT(R255X)和c.328CT (R110X)杂合突变,剩余病例未找到致病原因。R255X已在1个4代中国家系中报道,而R110X则见于2个中国家系,进一步验证发现这两个突变在各自的家系中是共分离的。2.成功构建pCMV-R255X-Flag突变质粒,瞬时转染进UACC903黑色素瘤细胞,发现突变的蛋白表达量明显减少,证实R255X突变的MITF基因编码的是一个截短型蛋白；免疫荧光实验发现,野生型MITF蛋白主要分布在细胞核中,而R255X突变蛋白除了在细胞核中分布外,也有相当大的一部分在细胞质中分布。3.运用GMR-GAL4、Da-GAL4驱动UAS-Mitf RNAi表达,其F1代的表型与对照组无明显差异。结论：1.MITF基因的R255X和R110X是中国人群WS2的突变热点,约75%的WS2病例未找到致病原因,提示存在未知的致病基因。2.R255X突变蛋白由于丧失了部分HLH、全部Zip及之后的全部区域,出现蛋白表达下降和异常的亚细胞分布,导致WS2的发生。完整的蛋白质和正确的亚细胞分布,对于MITF功能的行使具有重要意义。3.果蝇Mitf的表达具有时空上的特异性,需要更特异性的GAL4才能驱动其表达。
[Abstract]:Background: Waardenburg syndrome (Waardenburg Syndrome,WS) is a genetic syndrome characterized by hearing impairment and abnormal pigment distribution. WS can be divided into four types according to its clinical phenotype: WS1,WS2,WS3,WS4. WS is characterized by autosomal dominant inheritance and high genetic heterogeneity. WS can be divided into four types according to clinical phenotype. Among them, WS2 is the most common type. At present, it is found that MITF,SOX10 and SNAI2,MITF and SOX10 account for about 15% of all WS2 patients, and 70% of WS2 patients still do not find pathogenic factors, and the molecular mechanism of gene mutation leading to phenotype is not clear. Objective and methods: in order to further explore the internal relationship between genotype and phenotype in patients with WS2, the molecular mechanism of WS was discussed by the following schemes: 1. The WS2 family was collected, the detailed clinical data of each patient were collected, and the mutations in the exons of the related pathogenic gene MITF,SOX10, SNAI2 were screened. 2. For the R255X mutation of MITF gene, the eukaryotic expression plasmid pCMV-R255X-Flag, was constructed and the expression of mutant protein was verified by, Western Blot, and the subcellular distribution of mutant protein was detected by immunofluorescence. The phenotype of the first generation of Drosophila melanogaster was observed by crossing Mitf RNAi strain with GAL4 strain. Results: 1. A total of 3 WS2 families and 5 sporadic cases were collected. The c.763CT (R255X) and c.328CT (R110X) heterozygous mutations of MITF gene were found in two lines, and no cause was found in the remaining cases. R255X has been reported in a Chinese family of 4 generations, while R110X is found in two Chinese families. It is further confirmed that the two mutations are co-separated in their families. 2. The pCMV-R255X-Flag mutant plasmid was successfully constructed and transiently transferred into UACC903 melanoma cells. It was found that the expression of the mutant protein was significantly decreased, which confirmed that the MITF gene of R255X mutation encoded a truncated protein. Immunofluorescence assay showed that the wild type MITF protein was mainly distributed in the nucleus, while the R255X mutant protein was also distributed in the cytoplasm. Using GMR-GAL4,Da-GAL4 to drive the expression of UAS-Mitf RNAi, the phenotype of F1 generation was not significantly different from that of the control group. Conclusion: R255X and R110X of 1.MITF gene are the hot spots of WS2 mutation in Chinese population. About 75% of WS2 cases have no unknown pathogenic gene. 2.R255X mutant protein lost all Zip of HLH, and all regions after WS2, resulting in decreased protein expression and abnormal subcellular distribution, which led to the occurrence of R255X. Complete protein and correct subcellular distribution are of great significance for the exercise of MITF function. 3. The expression of Mitf in Drosophila melanogaster is space-time specific and requires more specific GAL4 to drive its expression.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R764;R3416

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