Regulation of ApoB Secretion in Inbred Mouse Strains

描述（由申请方提供）：载脂蛋白B（apo B）和低密度脂蛋白（LDL）的血浆水平升高与动脉粥样硬化性冠心病（CHD）的高风险相关，CHD是工业化国家死亡的主要原因。血浆载脂蛋白B水平升高和载脂蛋白B过度产生也是家族性混合型高脂血症（FCHL）的主要特征，FCHL是一般人群中的一种流行疾病。这种流行病的遗传基础是异质性和未知的。调节血浆载脂蛋白B水平的基因是促成FCHL表型的基因的逻辑候选者。载脂蛋白B是从肝脏分泌极低密度脂蛋白（VLDL）所必需的，是血浆VLDL和LDL的必需蛋白质成分。血浆载脂蛋白B水平部分受含载脂蛋白B的脂蛋白颗粒的分泌速率控制。使用人载脂蛋白B转基因小鼠模型（HuBTg），我们已经证明C57 BL/6（B6）和129/Sv（129）背景的HuBTg小鼠血浆载脂蛋白B水平不同，主要是由于肝脏载脂蛋白B分泌速率通过转录后调节所致。对两个小鼠品系的进一步遗传研究在染色体6和4上鉴定了两个新的数量性状位点（QTL），其对血浆载脂蛋白B水平具有主要影响。这些基因被命名为Apo B调节基因（Abrg）。我们的精细定位分析已经将Abrgl定位到染色体6上的约2.9Mb的间隔，并且将Abrg 2定位到染色体4上的12-cM的间隔。本发明的目标是鉴定和表征6号染色体上的Abrgl基因。具体目标是：1.调节血浆apo B水平的6号染色体上Abrgl的定位克隆。将采用几种平行的方法来鉴定Abrgl的候选基因。这些方法包括RT-PCR、定量实时PCR测定和DNA测序分析。还将设计研究以确定表达变体的等位基因变异对转录的影响。2.同源小鼠中Abrg基因的表征和Abrgl候选基因的功能研究。我们将通过体外和体内过表达研究对候选变异基因进行功能测试，包括Copsla基因（表达变体）。Abrgl基因的最佳候选物，Cops 7a或待鉴定的另一种基因，然后将通过敲入和/或敲除基因靶向技术进行验证。将设计研究以表征Abrgl基因的功能作用。总的来说，所鉴定的基因将是一种新的调节剂，影响载脂蛋白B的组装和分泌途径。作为apo B分泌速率的调节因子，该新基因将是FCHL患者亚群中apo B过度产生的基因之一的强有力候选者。它也将成为普通人群中高血压患者药物发现和药理学干预的潜在靶点。

DESCRIPTION (provided by applicant): Elevated plasma levels of apolipoprotein B (apo B) and low density lipoprotein (LDL) are associated with a higher risk for atherosclerotic coronary heart disease (CHD), a leading cause of mortality in the industrialized world. Elevated plasma apo B levels and overproduction of apo B are also major characteristics of familial combined hyperlipidemia (FCHL), a prevalent disorder in the general population. The genetic basis of this prevalent disease is heterogeneous and unknown. Genes that regulate plasma apo B levels are logical candidates for genes which contribute to the FCHL phenotype. Apo B is required for the secretion of very low density lipoproteins (VLDL) from the liver and is the mandatory protein constituent of both plasma VLDL and LDL. Plasma apo B levels are controlled, in part, by secretion rates of apo B-containing lipoprotein particles. Using the human apo B transgenic mouse model (HuBTg), we have shown that HuBTg mice of C57BL/6 (B6) and 129/Sv (129) background differ in their plasma apo B levels mainly due to hepatic apo B secretion rates via post-transcriptional regulation. Further genetic studies of two mouse strains identified two novel quantitative trait loci (QTL) on chromosomes 6 and 4 which have major effects on plasma apo B levels. These genes are designated Apo B regulatory genes (Abrg). Our fine-mapping analyses have localized the Abrgl to an interval of approximately 2.9 Mb on chromosome 6 and the Abrg2 to a 12-cM interval on chromosome 4. The goals of the current proposal are to identify and characterize the Abrgl gene on chromosome 6. The specific aims are: 1. Positional cloning of the Abrgl on chromosome 6 that regulates plasma apo B levels. Several parallel approaches will be taken to identify candidate genes for the Abrgl. These approaches include RT-PCR, quantitative real-time PCR assays, and DNA sequencing analysis. Studies will also be designed to determine the effects of allelic variations of expression variants on transcription. 2. Characterization of the Abrg genes in congenic mice and functional studies of the Abrgl candidate genes. We will perform functional tests on candidate variant genes, including the Copsla gene (an expression variant), by over expression studies in vitro and in vivo. The best candidate for the Abrgl gene, either Cops7a or another gene to be identified, will then be verified via knock-in and/or knock-out gene-targeting techniques. Studies will be designed to characterize the functional roles of the Abrgl gene. Overall, the gene identified will be a novel regulator affecting the pathways involved in the assembly and secretion of apo B containing lipoproteins. As a regulator of apo B secretion rates, this novel gene will be a strong candidate for one of the genes causative for overproduction of apo B in subsets of FCHL patients. It will also be a potential target for drug discovery and pharmacological intervention in hyperlipidemic patients in the general population.