Wilson病ATP7B基因突变功能验证及mRNA剪接异常突变机制研究

Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism characterized by excessive copper deposition in various organs, particularly the liver, kidney, brain, and cornea. WD is caused by germline mutations in the ATP7B gene. We have discovered three novel germline mutations in the ATP7B gene. It is unknown about the influences of those mutations on ATP7B function. The purpose of our study was to determine the pathogenicity of these three novel variants detected in WD patients in vitro..Exonic Splicing Enhancer and Silencer (ESE and ESS, respectively), sequences that substantially contribute to alternative splicing by regulating the splice site selection, play an important role of the correct splicing of precursor-mRNA. Surprisingly, an increasing number of examples reveal that exonic mutations in ESEs and ESSs sequences disrupt the binding of splicing factors to these sequences or generate new splice sites or regulatory elements, causing disease. In our previous study, we used a bioinformatics program to analyze all point mutations of the ATP7B gene we detected in 40 WD patients and identify five candidate mutations (three missense mutations and two synonymous mutations) that may induce exon skipping. On the basis of this bioinformatics data, we next want to perform splicing assay using minigenes and binding experiments in vitro.

Wilson病（WD）是一种以铜代谢障碍为特征的常染色体隐性遗传病，由ATP7B基因突变所致。我们在前期40例WD患儿ATP7B基因检测工作中发现3种新突变，它们对ATP7B蛋白功能的影响尚不清楚。此外，有研究显示当点突变位于外显子剪接调控序列区时，突变导致疾病的机理是与外显子的异常剪接相关的。我们在40例WD患儿中发现5种可能导致mRNA剪接异常的错义/同义突变，这些突变可能破坏外显子剪接调控元件（ESEs和ESSs），影响其与剪接调控因子的相互作用。本研究拟通过质粒构建、定点诱变和细胞转染等方法探讨ATP7B新发突变对蛋白功能的影响；构建ATP7B mini基因剪接报告系统，通过RT-PCR、qPCR和测序等验证外显子区错义/同义突变对mRNA剪接的影响，应用RNA蛋白质pull down等实验初步探讨mRNA异常剪接产生的机理，希望为WD的临床筛查、诊断和遗传咨询提供新的实验依据。

肝豆状核变性(Wilson disease，WD)，是一种由铜转运P 型ATP酶( ATP7B )基因突变所致的常染色体隐性遗传病，以原发性铜代谢障碍为特征。很多WD儿童期症状往往是隐匿的，临床诊断存在一定的困难。目前基因诊断常规用于WD的临床诊断，但是对于基因诊断工作中碰到的ATP7B基因同义突变，往往因为其不改变蛋白编码潜能而被忽视，但是很多研究显示同义突变也可以通过影响mRNA剪接而导致遗传病。本研究通过构建Mini-gene剪接报告分析系统，验证本了课题组在基因检测工作中遇到的多种错义/同义突变和Exac数据库中247种罕见的ATP7B基因同义突变对外显子组成型剪接的影响。.我们课题组基因检测工作中发现的ATP7基因突变c.2755C>G(R919G) 数据库将其分类为具有不确定意义的突变。生物信息学分析显示c.2755C>G(R919G)变异外显子12上的剪接增强子，mini-gene结果显示该变异导致外显子12跳跃。c.4014T>A起初被预测为同义突变，无致病性，但本研究证明其导致外显子19的跳跃。Human Splicing Finder预测该突变产生了ESS,可以与剪接抑制因子hnRNP1结合，进而抑制了剪接。.在ExAc数据库中筛选出自然人群携带的等位基因频率小于0.005的247种罕见同义突变，用Human Splicing Finder 等分析247种罕见同义突变对前体mRNA剪接的影响。经分析，其中57种罕见同义突变会影响剪接受体或供体，54种罕见同义突变会对ESSs基序产生影响，37种罕见同义突变会对ESEs基序产生影响。经体外Mini-gene剪接报告分析系统验证，11种同义突变会导致异常的外显子跳跃。.我们的研究表明与剪接调控元件（外显子剪接增强子破坏和沉默子）相关的错义突变/同义突变导致的mRNA剪接异常是ATP7B基因突变导致WD的重要机制之一。