Alprot综合征基因突变对Ⅳ型胶原结构及功能的影响

The study aimed to explore the influence of post-mutations in COL4A5 gene on the structure and function of a5(IV) chain, which will help to understand the pathogenesis of Alport syndrome (AS), and further to benefit the future work of gene therapy. The strategy of this study was to detect and analyze the a5(IV) chain transcripted and translated from the mutation COL4A5 gene of AS patient. Total RNA was isolated from the cultured fibroblast obtained from skin biopsy. The whole length of a5(IV) chain coding region was amplified by 5 cDNA fragments with RT-PCR. The cDNA fragment was detected and checked via electrophoresis first and then sequencing. Any fragment with mutation would be further analyzed and confirmed by the sequences of relevant genomic DNA fragment (usually exon plus partial flank intronic sequences). The main achievements included, (1) Many novel mutations of COL4A5 gene were detected in Chinese AS kindreds. The results revealed that the mutation gene did not "turn-off" its transcription and, analysis of cDNA of fibroblasts instead of genomic DNA is a efficient (with a detection rate of 90%), convenient and economic way to diagnose COL4A5gene mutations. (2) AS with mutations of both COL4A5 and COL4A6 was firstly diagnosed in Chinese population. Those kind of patients presented with or without leomyomatosis will depend on different mutations in COL4A6 gene. (3) It is the first time in China that the heterozygous status in AS females was demonstrated by analyzing COL4A5 gene and the expression of the mutated gene. The segmental distribution of a5(Ⅳ) chain in basement membranes, the mild clinical symptoms and the better prognosis in AS females would belong to the heterozygous COL4A5 gene. (4) For the first time in China, a COL4A5 gene mutation was detected in an X-linked AS boy with normal expressions of a3-5 (IV) chains in epidermal and glomerular basement membranes. The result has been published in several national and international academic medical journals, including the journal of Pediatric Nephrology authorized by International Pediatric Nephrology Association, and also communicated widely during scientific congress.

在明确了Alport综合征（AS）基因突变的基础上，拟进一步用原位、Northern杂交和免疫荧光学方法，从肾脏和皮肤组成的mRNA和蛋白表达水平检测并分析Ⅳ胶原6个α链之一的α5链基因COL4A5突变后,α5及其它α链在转录和翻译水平的变化和异常，期望由此为进一步进行基因治疗在Ⅳ型胶原的分子组成特性和功能改变方面提供一定的依据。