肥胖型多囊卵巢致病基因候选克隆及其致病机理的研究

The incidence of Polycystic Ovary Syndrome is about5- 6%, most of them are obesity, the genes which have been found correlating with obesity of PCOS are Ghrelin, adiponectin et.al. Whether only one or with other mutation area of Ghrelin gene contributing to the obesity of PCOS and its molecular pathogenesis need to be confirmed. In this study, relying on the large-scale PCOS genetic resource database, 500 PCOS cases are performed for mutation screening of Ghrelin by using DHPLC mutation screening platform. On the basis of the mutation 501G/C in the promoter area of Ghrelin gene, combined with new mutations to be identified in this study, we will explore the effects of the mutations of Ghrelin gene on the obesity of PCOS. We will sequence and colon the mutations gene of Ghrelin using the Miseq sequencing platform, then the plasmid carrier will be completed and which will be transfectioned to the HELA cells and then injected to the PCOS mouse model, a series of molecular pathogenesis experiments will be confirmed the mutations of Ghrelin gene correlation with the obesity of PCOS, which will provide a new theoretical basis for the genetic intervention and treatment.

多囊卵巢综合征(PCOS)在中国的发病率达5-6%,而肥胖型患者占PCOS的绝大部分,有关PCOS肥胖发病基因目前被发现的仅有Ghrelin(本研究小组发现),脂联素等,而Ghrelin是单独一个突变位点影响还是与其它突变位点连锁影响PCOS肥胖的发生,以及其影响的分子机制有待研究证实。故本研究小组依托①已经建立的大型PCOS遗传资源库，以500余例PCOS为研究对象，采用DHPLC突变/多态筛选平台对PCOS的Ghrelin基因外显子进行突变筛查，在已经发现的启动子区突变位点501G/C的基础上，结合新发现的突变位点进行连锁分析与PCOS肥胖的相关性；②采用Miseq技术平台对肥胖PCOS患者进行突变Ghrelin基因验证克隆；③构筑突变基因真核质粒表达载体，转染细胞并注射动物后进一步从分子机制验证Ghrelin突变基因参与PCOS肥胖的发生，为将来进行遗传学干预和治疗提供新的理论基础。

多囊卵巢综合征(Polycystic ovary syndrome，PCOS)是育龄期女性常见的内分泌代谢性疾病，其发病率在肥胖女性中越来越普遍，同时肥胖型PCOS女性存在更为严重的内分泌激素紊乱和代谢功能异常。肥胖和PCOS均为病因不明的异质性疾病，但认为二者均受环境因素和遗传因素的影响。本研究基于前期本课题组发现的PCOS肥胖相关基因Ghrelin501G/C突变位点的基础上，将收集的PCOS患者和正常人群的血样通过多重聚合酶链反应技术对Ghrelin基因全长位点靶向捕获后进行高通量测序，通过测序鉴定出24个相关位点，包括先前报道的外显子区域rs696217、rs34911341和启动子区域的rs26802、rs27647位点，结合这4个位点和内含子区域的rs35681、rs2075356六个位点进行关联性分析，通过分析发现位于第四内含子的rs35681位点可能与PCOS相关肥胖的发生有关，位点A等位基因增加了PCOS肥胖发生的风险。在连锁不平衡分析中，鉴定出6个基因座处于不完全连锁不平衡状态。此外，单倍型分析表明AAGCAA单倍型可能是PCOS易感性的原因。后期将rs35681位点通过CRISPR/CAS9基因编辑技术进行质粒构建，并通过转染敲入HELA细胞后测序发现rs35681位点同源序列套峰出现，然后将转染成功的细胞单克隆并进行稳转株构建。本研究从临床样本入手，通过关联分析证实了rs35681与PCOS肥胖的相关性，由于rs35681位点位于Ghrelin基因第四内含子，内含子SNP作用机制尚不明确，为后续研究内含子SNP作用机制奠定了基础。