肠易激综合征肠道线粒体全基因组单核苷酸多态性 (SNP)的筛选与鉴定

Irritable bowel syndrome (IBS) is one of the common digestive diseases in the outpatient department, of which the pathogenesis remains unclear. How genetic background play a role in IBS has been attracting much attention recently. It was noted that familial aggregation was common in patients with irritable bowel syndrome, especially with mothers and her children involved. As we know, mitochondrial genome is maternally inherited. So researchers are putting efforts to reveal the role of mitochondrial genome in IBS. Mitochondrial genome consists of control region, tRNA/sRNA genes as well as coding region of 13 genes for 13 subunits of enzymes such as ATP synthase and NADH dehydrogenase that involved in electron transmission during the ATP synthesis. A recent study revealed single nucleotide polymorphism (SNP) of control region of mitochondrial genome was associated with IBS with constipation (IBS-C). Whereas, how the SNP of mitochondrial DNA (mtDNA) was involved in the pathogenesis of IBS remains to be elucidated. What's more, we have no idea of all the SNPs of the whole mitochondrial genome in IBS. In a preliminary study, we studied the SNP of mitochondrial ATP 6 and 8 genes in IBS with diarrhea (IBS-D). Mitochondrial ATP 6 and 8 genes in intestine specimens were sequenced and polymorphisms derived. Our preliminary result showed patients with IBS-D have a higher incidence of mitochondrial ATP 6 and 8 polymorphisms than healthy subjects. All these evidences indicated that different SNP models exist in IBS-C and IBS-D, which may underlie the pathogenesis of IBS. In this project we aim to screen the SNPs of the whole mitochondrial genome, figure out those SNPs that may affect the function of the coding peptides in the electron transmission and ATP synthesis, subsequently identify the molecular candidates for the pathophysilogical study of IBS as well as for subtyping IBS.

肠易激综合征（IBS）是消化科一种常见病，其发病机制尚不清。遗传在IBS发病机制中的作用是目前研究热点之一。IBS有家庭内聚集现象，以母亲及其子女易患病，而线粒体基因组恰是母系遗传的，所以人们开始关注线粒体基因与IBS的关系。线粒体基因组含有编码区（编码ATP合成酶等电子传递链的13个亚基）、控制区等。近年来国外报道线粒体非编码区基因的单核苷酸多态性(SNP) 与便秘型IBS相关，但SNP如何参与IBS发病机制未见研究，而且也未有线粒体全基因组SNP的关联研究。我们前期工作中应用线粒体基因测序发现腹泻型IBS患者结肠线粒体ATP6和8基因SNP频率明显高于正常人。这都提示IBS亚型间线粒体SNP模式不同，可能参与了IBS发病机制。本课题拟在此基础上进一步筛选和分析不同亚型IBS线粒体全基因组的SNP模式，鉴定对电子传递链酶活性有影响的SNP，为IBS的病生机制、亚型分类提供分子依据。

一，项目背景: 肠易激综合征（irritable bowel syndrome, IBS）发病机制尚不明确。对于线粒体基因组在IBS发病机制中的作用知之甚少。因此本课题旨在研究腹泻型IBS（IBS-D）和便秘型IBS（IBS-C）患者的线粒体基因组单核苷酸基因多态性(single nucleotide polymorphisms；SNPs)，筛选及鉴定与IBS 相关的SNP位点。.二，研究方法: 所有研究对象均来自解放军总医院消化科门诊，根据临床诊断标准分成三组：IBS-D, IBS-C 和健康对照组。所有受试对象均行电子结肠镜检查并于退镜时在结肠活检，活检标本用常规方法提取基因组DNA，用特异性引物对线粒体基因组行PCR扩增及测序，然后和GenBank 标准序列（NC-012920）比对，并与人类线粒体基因组SNP 标准数据库MITOMAP ( www.mitomap.org ) 进行比较，分析并筛选出SNP 位点。对有意义的编码基因位点进一步鉴定，通过质粒转染大肠杆菌模型及ATP试剂盒进一步研究SNP位点对电子传递链酶功能的影响。其中选择大肠杆菌BL21菌株为功能实验的表达质粒载体菌株，并用标准株的基因序列为基础构建野生型及突变型质粒。 .三，重要结果及关键数据: 1. IBS-D 组、IBS-C 组和对照组在年龄及性别均无统计学差异； 2. 发现了一些以前未报道过的新SNP位点（表1）。3. 三组SNP 位点主要集中在高变区（I 和II），但三组SNP 位点在高变区的分布无显著差异（P=0.234）。 IBS-D 组、IBS-C 组和对照组的SNPs 数目为12.2±2.7、9.8±1.8 和9.9±2.1，IBS-D 组高于IBS-C 组和对照组（P=0.01），IBS-C 组和对照组无显著差异（P=0.91）；4.对全部SNP 位点逐个分析，发现线粒体基因组控制区的199 C位点在IBS-D 组、IBS-C 组两组有差异（P=0.03）。 .四，科学意义:线粒体基因组SNP频率增高与IBS-D密切相关，但与IBS-C不相关，提示线粒体基因组SNP在IBS-D发病机制中起重要作用。而且199位点基因多态性和IBS-D 发病风险有关，基因型199C 可能会降低IBS-D 的患病风险。这些研究结果为进一步研究IBS发病机制提供了新的分子标志及研究方向。